Antigen-binding molecules, the antigen-binding activity of which varies according to the concentration of compounds, and libraries of said molecules

ABSTRACT

An objective of the present invention is to provide target tissue-specific antigen-binding molecules, antigen-binding molecules whose antigen-binding activity varies depending on the concentration of an unnatural compound, libraries comprising a plurality of the antigen-binding molecules which are different from one another, pharmaceutical compositions comprising the antigen-binding molecules, methods of screening for the antigen-binding molecules, and methods for producing the antigen-binding molecules. The present inventors created antigen-binding domains whose antigen-binding activity varies depending on the concentration of a small molecule compound or antigen-binding molecules containing an antigen-binding domain, and libraries comprising a plurality of the antigen-binding domains which are different from one another or antigen-binding domains, and demonstrated that the above-noted objective could be achieved by using the libraries. Various diseases originating from target tissues can be treated in a target tissue-specific manner by using the antigen-binding molecules of the present invention.

TECHNICAL FIELD

The present invention relates to libraries of antigen-binding domains orantigen-binding molecules comprising an antigen-binding domain, of whichantigen-binding activity varies depending on the concentration of asmall molecule compound. The present invention also relates toantigen-binding domains or antigen-binding molecules comprising anantigen-binding domain, of which antigen-binding activity variesdepending on the concentration of an unnatural compound, productionmethods and screening methods for the antigen-binding molecules, andpharmaceutical compositions comprising the antigen-binding molecules.

BACKGROUND ART

Antibodies are drawing attention as pharmaceuticals as they are highlystable in plasma and have few side effects. In particular, a number ofIgG-type antibody pharmaceuticals are available on the market, and manyantibody pharmaceuticals are currently under development (Non-PatentDocuments 1 and 2).

As cancer therapeutic agents using antibody pharmaceuticals, Rituxanagainst a CD20 antigen, cetuximab against an EGFR antigen, herceptinagainst a HER2 antigen, and such have been approved so far (Non-PatentDocument 3). These antibody molecules bind to antigens expressed oncancer cells, and exhibit cytotoxic activity against cancer cells byADCC and such. Such cytotoxic activity by ADCC and etc. are known todepend on the number of antigens expressed on cells targeted by thetherapeutic antibodies (Non-Patent Document 4); therefore, highexpression level of the target antigen is preferable from the standpoint of the effects of the therapeutic antibodies. However, even if theantigen expression level is high, when antigens are expressed in normaltissues, cytotoxic activity mediated by ADCC etc. will be exertedagainst normal cells, and therefore side-effects will become a majorproblem. Therefore, antigens targeted by therapeutic antibodies used astherapeutic agents for cancer are preferably antigens specificallyexpressed in cancer cells. For example, antibody molecules against theEpCAM antigen which is known as a cancer antigen have been considered tobe promising as therapeutic agents for cancer. However, the EpCAMantigen is known to be expressed in the pancreas as well, and inpractice, administration of anti-EpCAM antibodies in clinical trials hasbeen reported to cause pancreatitis as a side-effect due to cytotoxicactivity towards the pancreas (Non-Patent Document 5).

Following the success of antibody pharmaceuticals that exert cytotoxicactivity by ADCC activity, a second generation of improved antibodymolecules that exert strong cytotoxic activity through enhancement ofADCC activity by removing fucose of N-type sugar chains in the nativehuman IgG1 Fc region (Non-Patent Document 6), enhancement of ADCCactivity by enhancing the binding toward FcγRIIIa by substitution ofamino acids in the native human IgG1 Fc region (Non-Patent Document 7),and such have been reported. As antibody pharmaceuticals that exertcytotoxic activity against cancer cells through a mechanism other thanthe above-mentioned ADCC activity mediated by NK cells, improvedantibody molecules that exert a stronger cytotoxic activity, such as anantibody-drug conjugate (ADC) in which an antibody is conjugated with adrug having potent cytotoxic activity (Non-Patent Document 8), and a lowmolecular weight antibody that exerts toxic activity against cancercells by recruiting T cells to cancer cells (Non-Patent Document 9),have been reported as well.

Such antibody molecules exerting a stronger cytotoxic activity can exertcytotoxic activity against cancer cells that do not have much antigenexpression, but on the other hand, they will exert similar cytotoxicactivity against normal tissues with low antigen expression. In fact, incomparison to cetuximab which is a natural human IgG1 against an EGFRantigen, EGFR-BiTE, which is a bispecific antibody against CD3 and EGFR,can exert a potent cytotoxic activity against cancer cells by recruitingT cells to cancer cells and exert antitumor effects. On the other hand,since EGFR is expressed also in normal tissues, when EGFR-BiTE isadministered to cynomolgus monkeys, serious side effects have appeared(Non-Patent Document 10). Furthermore, bivatuzumab mertansine, an ADCformed by linking mertansine to an antibody against CD44v6 which ishighly expressed in cancer cells, has been shown to cause severe skintoxicity and liver toxicity in clinical practice because CD44v6 isexpressed also in normal tissues (Non-Patent Document 11).

When antibodies that can exert a potent cytotoxic activity againstcancer cells having low antigen expression are used as such, the targetantigen needs to be expressed in a highly cancer-specific manner.However, since HER2 and EGFR, which are target antigens of herceptin andcetuximab, respectively, are also expressed in normal tissues, thenumber of cancer antigens expressed in a highly cancer-specific manneris thought to be limited. Therefore, while it is possible to strengthenthe cytotoxic activity against cancer, the side effects occurring due tocytotoxic actions against normal tissues may become problematic.

Furthermore, recently, ipilimumab which enhances tumor immunity byinhibiting CTLA4 which contributes to immunosuppression in cancer wasshown to prolong overall survival of metastatic melanoma (Non-PatentDocument 12). However, since ipilimumab inhibits CTLA4 systemically,while tumor immunity is enhanced, the emergence of autoimmunedisease-like severe side effects due to systemic activation of theimmune system is becoming a problem (Non-Patent Document 13).

On the other hand, as antibody pharmaceuticals against diseases besidescancer, antibody pharmaceuticals that exert therapeutic effects byinhibiting inflammatory cytokines in inflammatory/autoimmune diseasesare known (Non-Patent Document 14). For example, Remicade and Humirawhich target TNF, and Actemra which targets IL-6R exhibit hightherapeutic effects against rheumatoid arthritis, but on the other hand,systemic neutralization of these cytokines has led to the observation ofinfection as side effects (Non-Patent Document 15).

Various techniques have been developed as techniques that can be appliedto second-generation antibody pharmaceuticals. While techniques forimproving effector functions, antigen-binding ability, pharmacokinetics,and stability, or techniques for reducing immunogenic risks have beenreported (Non-Patent Document 16), there are hardly any reports ontechniques that enable target tissue-specific action of antibodypharmaceuticals to overcome such side effects. For example, regardinglesions such as cancer tissues and inflammatory tissues, pH-dependentantibodies that make use of the acidic pH condition at these targettissues have been reported (Patent Documents 1 and 2). However, thedecrease of pH (that is, increase in hydrogen ion concentration) incancer tissues and inflammatory tissues as compared to normal tissues isslight, and since it is difficult to produce antibodies that act bydetecting a slight increase in the concentration of hydrogen ions whichhave an extremely small molecular weight, and also because acidic pHconditions may be found in normal tissues such as osteoclastic boneresorption region or in tissues other than the lesion of interest, useof pH conditions as a lesion-specific environmental factor wasconsidered to face many challenges. On the other hand, methods forproducing antibodies that exert antigen-binding activity only after theyare cleaved by a protease expressed at lesion sites such as cancertissues and inflammatory tissues have been reported (Patent Document 3).However, since cleavage of antibodies by proteases is irreversible, whenthe antibodies that have been cleaved at the lesion site enter the bloodstream and return to normal tissues, they can bind to the antigens innormal tissues as well, and this is considered to be a problem.Furthermore, cancer specificity of such proteases is also thought tohave problems that need to be addressed. Therefore, techniques thatenable reversible action at sites of inflammation or cancer (lesionsites) without systemic action in normal tissues and blood for exertingdrug efficacy while avoiding side effects are not known. Further,methods for controlling antibody activities and pharmacological effectsby non-invasive administrations of exogenous compounds are not known.

PRIOR ART DOCUMENTS Patent Documents

-   [Patent document 1] WO 2003/105757-   [Patent document 2] WO 2012/033953-   [Patent document 3] WO 2010/081173

Non-Patent Documents

-   [Non-patent document 1] Monoclonal antibody successes in the clinic.    Janice M Reichert, Clark J Rosensweig, Laura B Faden & Matthew C    Dewitz, Nat. Biotechnol. (2005) 23, 1073-1078-   [Non-patent document 2] The therapeutic antibodies market to 2008.    Pavlou A K, Belsey M J., Eur. J. Pharm. Biopharm. (2005) 59 (3),    389-396-   [Non-patent document 3] Monoclonal antibodies: versatile platforms    for cancer immunotherapy. Weiner L M, Surana R, Wang S., Nat. Rev.    Immunol. (2010) 10 (5), 317-327-   [Non-patent document 4] Differential responses of human tumor cell    lines to anti-p185HER2 monoclonal antibodies. Lewis G D, Figari I,    Fendly B, Wong W L, Carter P, Gorman C, Shepard H M, Cancer Immunol.    Immunotherapy (1993) 37, 255-263-   [Non-patent document 5] ING-1, a monoclonal antibody targeting    Ep-CAM in patients with advanced adenocarcinomas. de Bono J S,    Tolcher A W, Forero A, Vanhove G F, Takimoto C, Bauer R J, Hammond L    A, Patnaik A, White M L, Shen S, Khazaeli M B, Rowinsky E K,    LoBuglio A F, Clin. Cancer Res. (2004) 10 (22), 7555-7565-   [Non-patent document 6] Non-fucosylated therapeutic antibodies as    next-generation therapeutic antibodies. Satoh M, Iida S, Shitara K.,    Expert Opin. Biol. Ther. (2006) 6 (11), 1161-1173-   [Non-patent document 7] Optimizing engagement of the immune system    by anti-tumor antibodies: an engineer's perspective. Desjarlais J R,    Lazar G A, Zhukovsky E A, Chu S Y., Drug Discov. Today (2007) 12    (21-22), 898-910-   [Non-patent document 8] Antibody-drug conjugates: targeted drug    delivery for cancer. Alley S C, Okeley N M, Senter P D., Curr. Opin.    Chem. Biol. (2010) 14 (4), 529-537-   [Non-patent document 9] BiTE: Teaching antibodies to engage T-cells    for cancer therapy. Baeuerle P A, Kufer P, Bargou R., Curr. Opin.    Mol. Ther. (2009) 11 (1), 22-30-   [Non-patent document 10] T cell-engaging BiTE antibodies specific    for EGFR potently eliminate KRAS- and BRAF-mutated colorectal cancer    cells. Lutterbuese R, Raum T, Kischel R, Hoffmann P, Mangold S,    Rattel B, Friedrich M, Thomas O, Lorenczewski G, Rau D, Schaller E,    Herrmann I, Wolf A, Urbig T, Baeuerle P A, Kufer P., Proc. Natl.    Acad. Sci. U.S.A. (2010) 107 (28), 12605-12610-   [Non-patent document 11] Phase I trial with the CD44v6-targeting    immunoconjugate bivatuzumab mertansine in head and neck squamous    cell carcinoma. Riechelmann H, Sauter A, Golze W, Hanft G, Schroen    C, Hoermann K, Erhardt T, Gronau S., Oral Oncol. (2008) 44 (9),    823-829-   [Non-patent document 12] Ipilimumab in the treatment of melanoma.    Trinh V A, Hwu W J., Expert Opin. Biol. Ther., (2012) Apr. 14    (doi:10.1517/14712598.2012.675325)-   [Non-patent document 13] IPILIMUMAB—A NOVEL IMMUNOMODULATING THERAPY    CAUSING AUTOIMMUNE HYPOPHYSITIS: A CASE REPORT AND REVIEW. Juszczak    A, Gupta A, Karavitaki N, Middleton M R, Grossman A., Eur. J.    Endocrinol. (2012) Apr. 10 (doi: 10.1530/EJE-12-0167)-   [Non-patent document 14] The Japanese experience with biologic    therapies for rheumatoid arthritis. Takeuchi T, Kameda H., Nat. Rev.    Rheumatol. (2010) 6 (11), 644-652-   [Non-patent document 15] Current evidence for the management of    rheumatoid arthritis with biological disease-modifying antirheumatic    drugs: a systematic literature review informing the EULAR    recommendations for the management of RA. Nam J L, Winthrop K L, van    Vollenhoven R F, Pavelka K, Valesini G, Hensor E M, Worthy G,    Landewe R, Smolen J S, Emery P, Buch M H., Ann. Rheum. Dis. (2010)    69 (6), 976-986-   [Non-patent document 16] Antibody engineering for the development of    therapeutic antibodies. Kim S J, Park Y, Hong H J., Mol.    Cells. (2005) 20 (1), 17-29

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

In view of the above-described background, if it is possible to obtainantibodies whose binding to a target antigen is regulated by theconcentration of a small molecule produced or specifically present in atarget tissue (hereinafter may be referred to as “small-molecule switchantibodies”), such antibodies will be very useful because they can actreversibly on lesions such as tumor sites and inflammatory sites, andside-effects can be avoided. Furthermore, if it is possible to obtainantibodies whose antigen binding is regulated by the concentration of anunnatural compound, such antibodies will be very useful since they canbe controlled by the administration of an exogenous compound thatactivates antibody activities and pharmacological actions at thelesions, or an exogenous compound that can be administerednon-invasively.

However, there are no reports that such antibodies have been obtained byconventional methods such as methods of immunizing non-human animalswith antigens, or methods of using a library of human-derived ornon-human animal-derived antibodies.

Therefore, there has been a strong desire to provide antibodies(small-molecule switch antibodies) whose binding to a discretionarytarget antigen is regulated by the concentration of a small moleculeproduced or specifically present in the target tissue or an unnaturalcompound, and methods for efficiently obtaining such antibodies in ashort period of time.

Means for Solving the Problems

The present inventors conducted dedicated studies to achieve theabove-described objectives. As a result, they generated antigen-bindingmolecules comprising an antigen-binding domain whose antigen-bindingactivity varies depending on the concentration of the targettissue-specific compound. Furthermore, the present inventors discoveredthat the antigen-binding molecules or pharmaceutical compositionscomprising the antigen-binding molecules are useful for treatingdiseases that originate from a target tissue, and that they are alsouseful for treatment of diseases originating from target tissues thatincludes administering the antigen-binding molecules. They alsodiscovered that the antigen-binding molecules are useful in theproduction of pharmaceuticals for treating diseases that originate fromtarget tissues.

The present inventors also successfully produced a library comprising aplurality of antigen-binding molecules having different sequences fromone another, wherein the molecules have an antigen-binding domain thatcomprises amino acid residues involved in binding with a small moleculethat may cause the antigen-binding activity of the antigen-bindingmolecule to vary according to differences in the in vivo environmentalfactors or depending on administration of an unnatural compound. Theyalso created methods for screening and producing the antigen-bindingmolecules using the library, and thereby completed the presentinvention.

The present invention is based on such findings, and specificallyincludes embodiments exemplified below.

Embodiment 1

A library that comprises mainly:

(i) a plurality of antigen-binding domains or antigen-binding moleculescomprising an antigen-binding domain, which have different sequencesfrom one another; or

(ii) nucleic acids that encode the plurality of antigen-binding domainsor antigen-binding molecules comprising an antigen-binding domain, whichhave different sequences from one another;

wherein the aforementioned antigen-binding molecule domains orantigen-binding molecules are antigen-binding domains or antigen-bindingmolecules comprising an antigen-binding domain whose antigen-bindingactivity varies depending on the concentration of a small moleculecompound.

Embodiment 2

The library of Embodiment [1], which is produced by a method comprisingthe steps of:

(a) identifying amino acid sites that fulfill any one or more of (i) to(iii) below in antigen-binding domains whose antigen-binding activityvaries depending on the concentration of a small molecule compound or inantigen-binding domains that have binding activity to a small moleculecompound:

(i) one or more amino acid sites that are not involved in the binding tothe small molecule compound;

(ii) one or more amino acid sites that show diversity of amino acidoccurrence frequency in the antibody repertoire of the animal species towhich the parent antigen-binding domain belongs; and

(iii) one or more amino acid sites that are not important for canonicalstructure formation; and

(b) designing a library that comprises nucleic acids encoding unmodifiedantigen-binding domains/molecules, and nucleic acids that encodeindividually a plurality of variants of the aforementionedantigen-binding domains or antigen-binding molecules comprising anantigen-binding domain which have different sequences from one anotherand have modifications at one or more of the amino acid sites identifiedin step (a).

Embodiment 3

The library of Embodiment [2] which is produced by a method comprisingthe steps of:

(a) identifying amino acid sites that fulfill any one or more of (i) to(iii) below in antigen-binding domains whose antigen-binding activityvaries depending on the concentration of a small molecule compound or inantigen-binding domains that have binding activity to a small moleculecompound:

(i) one or more amino acid sites that are not involved in the binding tothe small molecule compound;

(ii) one or more amino acid sites that show diversity of amino acidoccurrence frequency in the antibody repertoire of the animal species towhich the parent antigen-binding domain belongs; and

(iii) one or more amino acid sites that are not important for canonicalstructure formation;

(b) producing a plurality of variants of the aforementionedantigen-binding domains or antigen-binding molecules comprising anantigen-binding domain, which have different sequences from one anotherand have modifications at one or more of the amino acid sites identifiedin step (a);

(c) identifying one or more amino acid modifications that do notsubstantially change the binding activity of each of the aforementionedvariants to the small molecule compound; and

(d) producing a library comprising nucleic acids that encode unmodifiedantigen-binding domains/molecules, and nucleic acids that encode aplurality of variants of the aforementioned antigen-binding domains orantigen-binding molecules comprising an antigen-binding domain, whichhave different sequences from one another and have one or more of theamino acid modifications identified in step (c).

Embodiment 4

The library of Embodiment [1] produced by a method comprising the stepsof:

1) contacting a library comprising a plurality of antigen-bindingmolecules having binding activity to a small molecule compound with thesmall molecule compound; and

2) concentrating from the library, nucleic acids that encode a pluralityof variants of antigen-binding molecules having binding activity to thesmall molecule compound.

Embodiment 5

The library of Embodiment [4], wherein the aforementionedantigen-binding molecules are antigen-binding molecules that compriseheavy-chain variable regions and light-chain variable regions of anantibody, and wherein the library is produced by a method comprising anyone of the steps of:

1) designing a library by concentrating nucleic acids that encode aplurality of variants of antigen-binding molecules having bindingactivity to a small molecule compound from the library of Embodiment [4]which comprises nucleic acids encoding one or more variants produced bymodifying amino acids positioned in the heavy chain variable regions;

2) designing a library by concentrating nucleic acids that encode aplurality of variants of antigen-binding molecules having bindingactivity to a small molecule compound from the library of Embodiment [4]which comprises nucleic acids encoding one or more variants produced bymodifying amino acids positioned in the light chain variable regions;and

3) designing a library by combining the antigen-bindingmolecule-encoding nucleic acids concentrated from each of the variableregion libraries of steps 1) and 2).

Embodiment 6

The library of any one of Embodiments [1] to [5], wherein theaforementioned antigen-binding molecules are fusion polypeptides formedby fusing an antigen-binding domain with at least a portion of a viruscoat protein.

Embodiment 7

The library of any one of Embodiments [1] to [5], wherein theaforementioned antigen-binding molecules are antigen-binding moleculescomprising antibody heavy chains and light chains, and the libraryfurther comprises a step of designing a synthetic library of the heavychains and/or light chains.

Embodiment 8

The library of Embodiment [7], wherein the antibody heavy chains and/orlight chains comprise a germline-derived framework sequence.

Embodiment 9

The library of any one of Embodiments [1] to [8], wherein theaforementioned small molecule compound is a target tissue-specificcompound or an unnatural compound.

Embodiment 10

The library of any one of Embodiments [1] to [9], wherein theaforementioned target tissue is a cancer tissue or an inflammatorytissue.

Embodiment 11

The library of Embodiment [10], wherein the cancer tissue-specificcompound is at least one compound selected from the group consisting ofnucleosides that have a purine ring structure, amino acids and theirmetabolites, lipids and their metabolites, primary metabolites fromsugar metabolism, and nicotinamide and its metabolites.

Embodiment 12

The library of any one of Embodiments [1] to [11], wherein the smallmolecule compound is kynurenine, adenosine, adenosine monophosphate,adenosine diphosphate, or adenosine triphosphate.

Embodiment 13

The library of any one of Embodiments [1] to [12], wherein the aminoacid sites not involved in binding with the small molecule compound aresites other than any one or more of the amino acids selected from below:

H chain: 97, 100c, 101, 94, 95, 100d, 100e, 33, 50, 52, 56, 57, 58, 99,100, 100a, 54, 55 (Kabat Numbering); and

L chain: 49, 55, 95c, 96, 95a, 95b (Kabat Numbering).

Embodiment 14

A method for producing an antigen-binding molecule comprising anantigen-binding domain whose antigen-binding activity varies dependingon the concentration of a small molecule compound, which comprises thesteps of:

(a) contacting the library of any one of Embodiments [1] to [13] with anantigen in the absence of a small molecule compound;

(b) selecting an antigen-binding domain that does not bind to theantigen in step (a) above;

(c) contacting the antigen-binding domain selected in step (b) abovewith the antigen in the presence of the small molecule compound;

(d) selecting the antigen-binding domain that binds to the antigen instep (c) above;

(e) linking the polynucleotide that encodes the antigen-binding domainselected in step (d) above with a polynucleotide that encodes apolypeptide comprising an Fc region;

(f) culturing a cell introduced with a vector in which thepolynucleotide obtained in step (e) above is operably linked; and

(g) collecting the antigen-binding molecule from the culture solution ofthe cell cultured in step (f) above.

Embodiment 15

A method for producing an antigen-binding molecule comprising anantigen-binding domain whose antigen-binding activity varies dependingon the concentration of a small molecule compound, which comprises thesteps of:

(a) contacting the library of any one of Embodiments [1] to [13] with anantigen in the presence of a small molecule compound;

(b) collecting an antigen-binding domain by dissociating it using thesmall molecule compound at a lower concentration than in step (a) above;

(c) linking the polynucleotide that encodes the antigen-binding domaincollected in step (b) above with a polynucleotide that encodes apolypeptide comprising an Fc region;

(d) culturing a cell introduced with a vector in which thepolynucleotide obtained in step (c) above is operably linked; and

(e) collecting the antigen-binding molecule from the culture solution ofthe cell cultured in step (d) above.

Embodiment 16

The method of Embodiment [14] or [15] for producing an antigen-bindingmolecule that comprises an antigen-binding domain whose antigen-bindingactivity varies depending on the concentration of a small moleculecompound, which further comprises the steps of:

(a) contacting the library of any one of Embodiments [1] to [13] with asmall molecule compound; and

(b) selecting antigen-binding domains collected in step (a) above.

Embodiment 17

The method of any one of Embodiments [14] to [16] for producing anantigen-binding molecule, wherein the small molecule compound iskynurenine, adenosine, adenosine monophosphate, adenosine diphosphate,or adenosine triphosphate.

Embodiment 18

An antigen-binding molecule comprising an antigen-binding domain whoseantigen-binding activity varies depending on the concentration of anunnatural compound.

Embodiment 19

A pharmaceutical composition that comprises the antigen-binding moleculeof Embodiment [18].

Those skilled in the art will naturally understand that the presentinvention includes any combination of one or more embodiments describedabove, as long as it is not technically inconsistent with commontechnical knowledge of those skilled in the art.

Effects of the Invention

The antigen-binding domains or antigen-binding molecules comprising anantigen-binding domain of the present invention, whose antigen-bindingactivity varies depending on the concentration of a small moleculecompound, and pharmaceutical compositions comprising thereof do not actsystemically in the blood or in normal tissues; however, by actingreversibly at lesions such as cancers or inflamed sites in targettissues, they show drug efficacy while avoiding side-effects, and cantreat diseases originated at the target tissues.

Furthermore, by using libraries of the present invention comprising aplurality of antigen-binding domains or antigen-binding molecules thatcomprise an antigen-binding domain and have different sequences from oneanother, and whose antigen-binding activity varies depending on theconcentration of a small molecule compound, various antigen-bindingmolecules useful for treating tissue-specific diseases such as thosedescribed above can be obtained efficiently in a short period of time.

In an embodiment of the libraries of the present invention, amino acidsites in antigen-binding molecule domains that are not involved in thebinding to a small molecule compound are identified, and a library isdesigned to comprise nucleic acids that encode antigen-binding domainshaving different sequences from one another so that the amino acids atthe identified sites become one to several types of amino acids. Thisprovides a library that can yield antigen-binding molecules whoseantigen-binding ability varies in the presence of the compound moreefficiently than using a library of antibodies derived from humans ornon-human animals or a method of immunizing non-human mammals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that a small-molecule-switch antibody does not bind toantigens in a normal environment where the small molecules are notpresent, but binds to the antigens in the target tissue where the smallmolecules are present at a high concentration.

FIG. 2 shows that the small molecule functions as a switch by fittingbetween the anti-small-molecule antibody and the antigen. If the smallmolecule is absent, the antibody-antigen interaction is insufficient andthe antibody cannot bind to the antigen, but if the small molecule ispresent, the antibody can bind to the antigen by having the smallmolecule placed between the antibody and the antigen.

FIG. 3 shows the structure of 2′-Adenosine-PEG-peptide which is anadenosine analog used for immunization of rabbits.

FIG. 4 shows the structure of 5′-Adenosine-PEG-peptide which is anadenosine analog used for immunization of rabbits.

FIG. 5 shows the structure of 2′-Adenosine-PEG-biotin formed bysubstituting biotin for the peptide portion of the adenosine analog usedfor immunization of rabbits.

FIG. 6 shows the structure of 5′-Adenosine-PEG-biotin formed bysubstituting biotin for the peptide portion of the adenosine analog usedfor immunization of rabbits.

FIG. 7 shows results of comparing the 2′-Adenosine-PEG-Biotin-bindingactivities of the individual antibodies obtained by rabbit B cellscloning. The vertical axis shows the value (N_binding_100) obtained bydividing the amount of each antibody bound in the interaction with2′-Adenosine-PEG-biotin by the capture level (RU) of each antibody, andthe horizontal axis shows the value (N_stability_100) obtained bydividing the value obtained 60 seconds after dissociation of2′-Adenosine-PEG-biotin from each antibody after its interaction with2′-Adenosine-PEG-biotin by the capture level (RU) of each antibody.

FIG. 8A shows sensorgrams of surface plasmon resonance-based analysisdemonstrating that clone SMB0002 binds to (interacts with) adenosine.The sensorgrams show interactions between SMB0002 and adenosine at 100(in duplicate), 50, 25, 12.5, 6.25, and 3.13 nM in order from the top.

FIG. 8B shows sensorgrams of surface plasmon resonance-based analysisdemonstrating that clone SMB0002 binds to (interacts with) ATP. Thesensorgrams show interactions between SMB0002 and ATP at 5000, 1250,313, and 78.1 nM in order from the top.

FIG. 9 shows results of competitive ELISA demonstrating that cloneSMB0002 binds to adenosine and ATP.

FIG. 10A shows sensorgrams of surface plasmon resonance-based analysisdemonstrating that clone SMB0002 binds to (interacts with) AMP. Thesensorgrams show interactions between SMB0002 and AMP at 500, 250 (induplicate), 125, 62.5, 31.3, 15.6, and 7.81 μM in order from the top.

FIG. 10B shows sensorgrams of surface plasmon resonance-based analysisdemonstrating that clone SMB0002 binds to (interacts with) ADP. Thesensorgrams show interactions between SMB0002 and ADP at 2000, 1000 (induplicate), 500, 250, 125, 62.5, and 31.3 μM in order from the top.

FIG. 11A shows the mode of binding between the SMB0002 antibody and theadenine ring portion of adenosine. In the figure, thick lines show the Hchain and thin lines show the L chain of the antibody, and adenosine isshown by a ball-and-stick model. The amino acid residues at distances of3.8 Å or less from the adenine ring are shown by a stick model. Thedashed lines show hydrogen bonds having a distance of 3.2 Å or lessbetween the antibody and the adenine ring portion.

FIG. 11B shows the mode of binding between the SMB0002 antibody and theribose portion of adenosine. In the figure, thick lines show the H chainand thin lines show the L chain of the antibody, and adenosine is shownby a ball-and-stick model. The amino acid residues at distances of 3.8 Åor less from the ribose portion are shown by a stick model. The dashedlines show hydrogen bonds having a distance of 3.2 Å or less between theantibody and the ribose portion. The area within the dotted lines showsthe region of the predicted presence of the phosphate group when boundto AMP.

FIG. 12 shows sensorgrams of surface plasmon resonance-based analysisdemonstrating that humanized SMB0002 binds to (interacts with)adenosine. The sensorgrams show interactions between humanized SMB0002and adenosine at 200, 100, 50 (in duplicate), 25, 12.5, 6.25, and 3.125nM in order from the top.

FIG. 13 shows sensorgrams of surface plasmon resonance-based analysisdemonstrating that humanized SMB0002 binds to (interacts with) AMP. Thesensorgrams show interactions between humanized SMB0002 and AMP at 500,250, 125 (in duplicate), 62.5, 31.3, 15.6, and 7.8 μM in order from thetop.

FIG. 14 shows sensorgrams of surface plasmon resonance-based analysisdemonstrating that humanized SMB0002 binds to (interacts with) ADP. Thesensorgrams show interactions between humanized SMB0002 and ADP at 1000(in duplicate), 500, 250, 125, and 62.5 μM in order from the top.

FIG. 15 shows sensorgrams of surface plasmon resonance-based analysisdemonstrating that humanized SMB0002 binds to (interacts with) ATP. Thesensorgrams show interactions between humanized SMB0002 and ATP at 1000(in duplicate), 500, 250, 125, and 62.5 μM in order from the top.

FIG. 16 is a figure showing the result of ELISA for binding of clone6RNMSC1-2_F02 to human IL-6R. The vertical axis shows the absorbancevalues which assess the binding activity of the antibody to human IL-6Rin the presence or absence of each small molecule.

FIG. 17 is a figure showing the result of ELISA for binding of clone6RNMSC1-3_G02 to human IL-6R. The vertical axis shows the absorbancevalues which assess the binding activity of the antibody to human IL-6Rin the presence or absence of each small molecule.

FIG. 18 is a figure showing the result of ELISA for binding of anantibody to human IL-6R. The vertical axis shows the absorbance valueswhich assess the binding activity of the antibody to human IL-6R in thepresence or absence of each amino acid or amino acid metabolite.

FIG. 19 presents sensorgrams showing the interaction between6RNMSC1-2_F02 and 1 μmol/L IL-6R in the presence of 100 μmol/Lkynurenine, in the presence of 10 mmol/L ATP, and in the absence ofkynurenine and ATP. The solid line indicates the interaction in thepresence of kynurenine, the dotted line indicates the interaction in thepresence of ATP, and the dashed line indicates the interaction in theirabsence.

FIG. 20 is a graph obtained by allowing 6RNMSC1-2_F02 to interact withIL-6R immobilized on Sensor chip CM5 in the presence of 100 μmol/Lkynurenine, and then observing the dissociation of 6RNMSC1-2_F02 fromIL-6R under conditions of a buffer containing 100 μmol/L kynurenine or abuffer that does not contain kynurenine. In the figure, the verticalaxis shows values normalized by defining the amount of 6RNMSC1-2_F02bound in the presence of 100 μmol/L kynurenine as 100, and thehorizontal axis shows the passage of time (in seconds) from the start ofthe interaction. The solid line shows the dissociation of 6RNMSC1-2_F02from IL-6R in the presence of kynurenine, and the dotted line shows thedissociation of 6RNMSC1-2_F02 from IL-6R in the absence of kynurenine.

FIG. 21 is a graph produced by allowing 5 μg/L of 6RNMSC1-2_F02 tointeract as an analyte for 180 seconds, and assessing the response toIL-6R immobilized onto Sensor chip CM5. The vertical axis shows changein the response (RU) before and after 6RNMSC1-2_F02 interaction, and thehorizontal axis shows the concentration (μmol/L) of kynurenine containedin the solution.

FIG. 22 shows sensorgrams of surface plasmon resonance-based analysisdemonstrating that clone 6RNMSC1-2_F02 binds to (interacts with)kynurenine. The sensorgrams show interactions between 6RNMSC1-2_F02 andkynurenine at 1.25, 0.625, 0.313, 0.156, 0.078, and 0.039 mM in orderfrom the top. The kinetic parameters are ka=709 (l/s), kd=0.17 (l/s),and KD=0.239 (mmol/L).

FIG. 23 shows sensorgrams of surface plasmon resonance-based analysisdemonstrating that clone 6RNMSC1-2_F02 binds to (interacts with)3-hydroxy-DL-kynurenine. The sensorgrams show interactions between6RNMSC1-2_F02 and 3-hydroxy-DL-kynurenine at 0.625, 0.313, 0.156, 0.078,and 0.039 mM in order from the top.

FIG. 24 shows sensorgrams of surface plasmon resonance-based analysisdemonstrating that clone 6RNMSC1-2_F02 binds to (interacts with) thecompound RO0635389-000-001. The sensorgrams show interactions between6RNMSC1-2_F02 and the compound RO0635389-000-001 at 0.625, 0.313, and0.156 mM in order from the top.

FIG. 25 shows sensorgrams of surface plasmon resonance-based analysisdemonstrating that clone 6RNMSC1-2_F02 binds to (interacts with) thecompound RO0635390-000-001. The sensorgrams show interactions between6RNMSC1-2_F02 and the compound RO0635390-000-001 at 0.625, 0.313, and0.156 mM in order from the top.

FIG. 26 shows Octet sensorgrams demonstrating that the binding(interaction) of clone 6RNMSC1-2_F02 with IL6R varies depending on thepresence (solid line) or absence (dashed line) of kynurenine. Thevertical axis shows the response to IL6R.

FIG. 27 shows Octet sensorgrams demonstrating that the binding(interaction) of clone 6RNMSC1-2_F02 with IL6R varies depending on thepresence (solid line) or absence (dashed line) of3-hydroxy-DL-kynurenine. The vertical axis shows the response to IL6R.

FIG. 28 shows Octet sensorgrams demonstrating that the binding(interaction) of clone 6RNMSC1-2_F02 with IL6R varies depending on thepresence (solid line) or absence (dashed line) of the compoundRO0635389-000-001. The vertical axis shows the response to IL6R.

FIG. 29 shows Octet sensorgrams demonstrating that the binding(interaction) of clone 6RNMSC1-2_F02 with IL6R varies depending on thepresence (solid line) or absence (dashed line) of the compoundRO0635390-000-001. The vertical axis shows the response to IL6R.

FIG. 30 shows the mode of binding between the 6RNMSC1-2_F02 Fab fragmentand kynurenine. In the figure, thick lines show the H chain and thinlines show the L chain of the antibody, and kynurenine is shown by aball-and-stick model. The amino acid residues at distances of 3.8 Å orless from kynurenine are shown by a stick model. The dashed linesindicate hydrogen bonds or electrostatic interactions having a distanceof 3.3 Å or less between the antibody and kynurenine.

FIG. 31 shows sensorgrams of surface plasmon resonance-based analysisdemonstrating that the H49Y variant of clone 6RNMSC1-2_F02 binds to(interacts with) kynurenine. The sensorgrams show interactions between6RNMSC1-2_F02H49Y and kynurenine at 1.25, 0.625, 0.313, 0.156, 0.078,and 0.039 mM in order from the top. The kinetic parameters are ka=2543(l/s), kd=0.24 (l/s), KD=0.095 (mmol/L).

FIG. 32 shows sensorgrams of surface plasmon resonance-based analysisdemonstrating that clone F02h011/F02l003, which is produced byintroducing mutations into the framework sequence of 6RNMSC1-2_F02 torestore the germline sequence, binds to (interacts with) kynurenine. Thesensorgrams show the interactions between F02h011/F02l003 and kynurenineat 1000, 500, 250, 125, and 62.5 μM in order from the top.

FIG. 33 shows sensorgrams of surface plasmon resonance-based analysisdemonstrating that clone F02h011/F02l098, which is produced byintroducing modifications that enhance kynurenine binding intoF02h011/F02l003, binds to (interacts with) kynurenine. The sensorgramsshow interactions between F02h011/F02l098 and kynurenine at 500, 250,125, 62.5, 31.3, and 15.6 μM in order from the top.

FIG. 34 shows the mode of binding between the 6RNMSC1-2_F02 Fab fragmentand kynurenine. In the figure, thick black lines show the heavy chainand thin grey lines show the light chain of the antibody, and kynurenineis shown by a ball-and-stick model. The amino acid residues at adistance of 4.2 Å or less from kynurenine are shown by a stick model.

FIG. 35 shows the mode of binding between the 6RNMSC1-2_F02 Fab fragmentand kynurenine. In the figure, thick black lines show the heavy chainand thin grey lines show the light chain of the antibody, and kynurenineis shown by a ball-and-stick model. The light chain Ser56 (Kabatnumbering) is shown by a stick model. The dashed line and the number onthe dashed line show the shortest distance between the non-hydrogenatoms of light chain Ser56 and kynurenine.

FIG. 36 shows the mode of binding between the 6RNMSC1-2_F02 Fab fragmentand kynurenine. In the figure, thick black lines show the heavy chainand thin grey lines show the light chain of the antibody, and kynurenineis shown by a ball-and-stick model. The heavy chain Gly50 and the lightchain Asp28 (Kabat numbering) are shown by a stick model.

FIG. 37 is a graph showing the level of binding (binding response (RU))when 1 μM of each clone was interacted with IL-6R immobilized on Sensorchip CM5 for 120 seconds in the presence or absence of each of the smallmolecules at 1 mM.

FIG. 38 is a graph showing the level of binding (binding response (RU))when 10 μg/mL of each clone was interacted with IL-6R immobilized onOctet sensors for 120 seconds in the presence or absence of each of thesmall molecules at 1 mM.

FIG. 39 shows results of ELISA performed on clones obtained from theVer. A kynurenine library, 6RFHm12-4_040, 6RFHm12-4_078, 6RFHm14-4_087,6RFHm14-4_093, 6RFHm17-4_006, and 6RFHm17-4_010, against hIL-6R underthe respective conditions. 6RNMSC1-2_F02 was used as the positivecontrol. The vertical axis shows the absorbance values for assessing thehIL-6-binding activity of the antibodies. Details of the respectiveconditions are shown in Table 38.

FIG. 40 shows results of ELISA performed on clones obtained from theVer. A kynurenine library, hIAFHm12-4_018, hIAFHm12-4_061,hIAFHm14-4_001, hIAFHm14-4_041, hIAFHm17-4_026, and hIAFHm17-4_072,against hIgA-Fc under the respective conditions. The vertical axis showsthe absorbance values for assessing the hIgA-Fc-binding activity of theantibodies. Details of the respective conditions are shown in Table 41.

FIG. 41 shows results of ELISA performed on clones obtained from theVer. A kynurenine library, I6FHm12-4_068, I6FHm12-4_094, I6FHm14-4_007,I6FHm14-4_030, I6FHm17-4_016, and I6FHm17-4_036, against hIL-6 under therespective conditions. The vertical axis shows the absorbance value forassessing the hIL-6-binding activity of the antibodies. Details of therespective conditions are shown in Table 44.

FIG. 42 is a graph that assesses the ability of ATP to inhibit bindingof ATNLSA1-4_D12 to the biotin-labeled antigen (a mixture of5′-Adenosine-PEG-biotin and ATP-PEG-biotin).

FIG. 43 is a figure for showing the concept of a rationally designedantibody library that can yield small-molecule-switch antibodies againstany antigen, wherein the library is made from antibody variable regionportions that are in contact with the antigen, and the small molecule ispositioned between the antibodies and the antigen as a switch.

FIG. 44 is a figure showing results of ELISA performed on cloneI6RLSA1-6_011, which was obtained from the rationally designed antibodylibrary using ATP/Adenosine-binding antibodies as the template, againsthuman IL-6 in the presence or absence of ATP and adenosine at 10 mM. Thevertical axis shows the absorbance value for evaluating the humanIL-6-binding activity of the antibody. The clone that shows humanIL-6-binding activity regardless of the presence or absence of a smallmolecule, which was obtained from the rationally designed antibodylibrary, was used as the positive control. M13KO7 Helper Phage was usedas the negative control.

FIG. 45 is a figure showing results of ELISA performed on clones6RRLSA1-6_037 and 6RRLSA1-6_045, which were obtained from the rationallydesigned antibody library using ATP/Adenosine-binding antibodies as thetemplate, against human IL-6 receptor in the presence or absence of ATPand adenosine at 10 mM. The vertical axis shows the absorbance value forevaluating the binding activity of the antibodies to the human IL-6receptor. M13KO7 Helper Phage was used as the negative control (shown asnega in the figure).

FIG. 46 is a figure showing the result of ELISA performed on cloneHSADSA1-6_020 obtained from the rationally designed antibody library,which library uses as a template antibodies that bind ATP/Adenosine,against HSA in the presence or absence of ATP and adenosine at 10 mM.The vertical axis shows the absorbance value which evaluates bindingactivity of the antibody to HSA. A clone obtained from the rationallydesigned antibody library and showing binding activity toward HSAregardless of the presence of small molecules was used as the positivecontrol. M13KO7 Helper Phage was used as the negative control.

MODE FOR CARRYING OUT THE INVENTION

The definitions and detailed description below are provided tofacilitate understanding of the present invention illustrated herein.

Amino Acids

Herein, amino acids are described by one- or three-letter codes or both,for example, Ala/A, Leu/L, Arg/R, Lys/K, Asn/N, Met/M, Asp/D, Phe/F,Cys/C, Pro/P, Gln/Q, Ser/S, Glu/E, Thr/T, Gly/G, Trp/W, His/H, Tyr/Y,Ile/I, or Val/V.

Alteration of Amino Acids

For amino acid alteration in the amino acid sequence of anantigen-binding molecule, known methods such as site-directedmutagenesis methods (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985)82, 488-492)) and overlap extension PCR may be appropriately employed.Furthermore, several known methods may also be employed as amino acidalteration methods for substitution to unnatural amino acids (Annu. Rev.Biophys. Biomol. Struct. (2006) 35, 225-249; and Proc. Natl. Acad. Sci.U.S.A. (2003) 100 (11), 6353-6357). For example, it is suitable to use acell-free translation system (Clover Direct (Protein Express))containing a tRNA which has an unnatural amino acid bound to acomplementary amber suppressor tRNA of one of the stop codons, the UAGcodon (amber codon).

In the present specification, the meaning of the term “and/or” whendescribing the site of amino acid alteration includes every combinationwhere “and” and “or” are suitably combined. Specifically, for example,“the amino acids at positions 33, 55, and/or 96 are substituted”includes the following variation of amino acid alterations:

amino acid(s) at (a) position 33, (b) position 55, (c) position 96, (d)positions 33 and 55, (e) positions 33 and 96, (f) positions 55 and 96,and (g) positions 33, 55, and 96.

Furthermore, herein, as an expression showing alteration of amino acids,an expression that shows before and after a number indicating a specificposition, one-letter or three-letter codes for amino acids before andafter alteration, respectively, may be used appropriately. For example,the alteration N100bL or Asn100bLeu used when substituting an amino acidcontained in an antibody variable region indicates substitution of Asnat position 100b (according to Kabat numbering) with Leu. That is, thenumber shows the amino acid position according to Kabat numbering, theone-letter or three-letter amino-acid code written before the numbershows the amino acid before substitution, and the one-letter orthree-letter amino-acid code written after the number shows the aminoacid after substitution. Similarly the alteration P238D or Pro238Aspused when substituting an amino acid of the Fc region contained in anantibody constant region indicates substitution of Pro at position 238(according to EU numbering) with Asp. That is, the number shows theamino acid position according to EU numbering, the one-letter orthree-letter amino-acid code written before the number shows the aminoacid before substitution, and the one-letter or three-letter amino-acidcode written after the number shows the amino acid after substitution.

Antigens

Herein, “antigens” are not particularly limited in their structure, aslong as they comprise epitopes to which antigen-binding domains bind. Inother words, antigens can be inorganic or organic substances. Otherantigens include, for example, the molecules below: 17-IA, 4-1BB, 4Dc,6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE,ACE-2, activin, activin A, activin AB, activin B, activin C, activinRIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB,ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAMS, ADAMS, ADAMTS,ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7,alpha-1-antitrypsin, alpha-V/beta-1 antagonist, ANG, Ang, APAF-1, APE,APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrialnatriuretic peptide, av/b3 integrin, Axl, b2M, B7-1, B7-2, B7-H,B-lymphocyte stimulating factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R,Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik,BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR,BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP,b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA,BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8,calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer associatedantigen, cathepsin A, cathepsin B, cathepsin C/DPPI, cathepsin D,cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S,cathepsin V, cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12,CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21,CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6,CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5,CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8,CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20,CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54,CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123,CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR,cGMP, CINC, Botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC,CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4,PD1, PDL1, LAG3, TIM3, galectin-9, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2,CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12,CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5,CXCR6, cytokeratin tumor associated antigen, DAN, DCC, DcR3, DC-SIGN,complement regulatory factor (Decay accelerating factor), des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV/CD26,Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN,ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM,ephrin B2/EphB4, EPO, ERCC, E-selectin, ET-1, factor IIa, factor VII,factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcR1,FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin,FL, FLIP, Flt-3, Flt-4, follicle stimulating hormone, fractalkine, FZD1,FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6,GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14,CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8(myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1,GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut4, glycoproteinIIb/IIIa (GPIIb/IIIa), GM-CSF, gp130, gp72, GRO, growth hormonereleasing hormone, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gBenvelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL,hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2,Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus(HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weightmelanoma-associated antigen (HMW-MAA), HIV gp120, HIV MB gp120 V3 loop,HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, humancytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309, TAP,ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGFbinding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2,IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12,IL-13, IL-15, IL-18, IL-18R, IL-21, IL-23, IL-27, interferon(INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain,insulin B chain, insulin-like growth factor1, integrin alpha2, integrinalpha3, integrin alpha4, integrin alpha4/beta1, integrin alpha4/beta7,integrin alpha5 (alpha V), integrin alpha5/beta1, integrin alpha5/beta3,integrin alpha6, integrin beta1, integrin beta2, interferon gamma,IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1,kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocytegrowth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1,latent TGF-1 bp1, LBP, LDGF, LECT2, lefty, Lewis-Y antigen, Lewis-Yassociated antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX,LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface,luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG,MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES,MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK,MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15,MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin(Muc1), MUC18, Mullerian-inhibiting substance, Mug, MuSK, NAIP, NAP,NCAD, N-C adherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4,or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO,NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150,p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD,P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2,PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), P1GF, PLP, PP14,proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specificmembrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL,RANTES, RANTES, relaxin A chain, relaxin B chain, renin, respiratorysyncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid factor, RLIP76, RPA2,RSK, S100, SCF/KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR,SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II,TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3,T-cell receptor (for example, T-cell receptor alpha/beta), TdT, TECK,TEM1, TEM5, TEM7, TEM8, TERT, testis PLAP-like alkaline phosphatase,TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific, TGF-betaRI(ALK-5), TGF-betaRII, TGF-betaRIIb, TGF-betaRIII, TGF-beta1, TGF-beta2,TGF-beta3, TGF-beta4, TGF-beta5, thrombin, thymus Ck-1,thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2,Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alphabeta, TNF-beta2, TNFc, TNF-RI,TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5,KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID),TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R),TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI),TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16(NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROYTAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60),TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNFRIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50),TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6),TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25(DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand,TL2), TNFSF11 (TRANCE/RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1,THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A/VEGI),TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-a Conectin, DIF,TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4(OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3,TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE,transferrin receptor, TRF, Trk, TROP-2, TLR1 (Toll-like receptor 1),TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TSG, TSLP, tumorassociated antigen CA125, tumor associated antigen expressing Lewis-Yassociated carbohydrates, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase,VCAM, VCAM-1, VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF,VEGFR, VEGFR-3 (flt-4), VEGI, VIM, virus antigen, VLA, VLA-1, VLA-4, VNRintegrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3,WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A,WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1,XEDAR, XIAP, XPD, HMGB1, IgA, Aβ, CD81, CD97, CD98, DDR1, DKK1, EREG,Hsp90, IL-17/IL-17R, IL-20/IL-20R, oxidized LDL, PCSK9, prekallikrein,RON, TMEM16F, SOD1, Chromogranin A, Chromogranin B, tau, VAP1, highmolecular weight kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3,Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, C1r, C1s,C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9,factor B, factor D, factor H, properdin, sclerostin, fibrinogen, fibrin,prothrombin, thrombin, tissue factor, factor V, factor Va, factor VII,factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X,factor Xa, factor XI, factor XIa, factor XII, factor XIIa, factor XIII,factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA,plasminogen, plasmin, PAI-1, PAI-2, GPC3, Syndecan-1, Syndecan-2,Syndecan-3, Syndecan-4, LPA, and S1P; and receptors for hormone andgrowth factors. Preferred antigens are antigens that are expressed incancer cells, immune cells, stromal cells, or such present in cancertissues or inflammatory tissues.

While receptors are recited as examples of the above-mentioned antigens,when these receptors exist in soluble forms in biological fluids, theymay also be used as antigens that bind to the antigen-binding moleculeof the present invention, which contains an antigen-binding domain whoseantigen-binding activity varies depending on the concentration of thesmall molecule compound (e.g., target tissue-specific compound). Anexample of a non-limiting embodiment of such a soluble receptor is thesoluble IL-6R, which is a protein consisting of the amino acids atpositions 1 to 357 in the IL-6R polypeptide sequence of SEQ ID NO: 1 asdescribed in Mullberg et al. (J. Immunol. (1994) 152 (10), 4958-4968).

Membrane-type molecules expressed on cell membranes and solublemolecules secreted from cells to the outside of the cells are includedin the examples of the above-mentioned antigens. When theantigen-binding molecule of the present invention, which contains anantigen-binding domain whose antigen-binding activity varies dependingon the concentration of the target tissue-specific compound, binds to asoluble molecule secreted from cells, it is preferable that theantigen-binding molecule has neutralizing activity as described later.

The fluids in which the soluble molecules exist are not limited, and thesoluble molecules may exist in biological fluids, or more specificallyin all fluids filling the space between tissues and cells or vessels inorganisms. In a non-limiting embodiment, the soluble molecules to whichantigen-binding molecules of the present invention bind may be presentin the extracellular fluid. In vertebrates, extracellular fluid is ageneral term for plasma, interstitial fluid, lymph, compact connectivetissue, cerebrospinal fluid, spinal fluid, puncture fluid, synovialfluid, or such components in the bone and cartilage, alveolar fluid(bronchoalveolar lavage fluid), peritoneal fluid, pleural fluid,pericardial effusion, cyst fluid, aqueous humor (hydatoid), or suchtranscellular fluids (various fluids in the glandular cavities andfluids in the digestive tract cavity and other body cavity fluidsproduced as a result of active transport/secretory activities of cells).

When an antigen-binding molecule of the present invention comprising anantigen-binding domain whose antigen-binding activity varies dependingon the concentration of a small molecule compound (e.g., targettissue-specific compound) binds to a membrane-type molecule expressed ona cell membrane, suitable examples of the antigen-binding moleculeinclude antigen-binding molecules which have cytotoxic activity, bind toa cytotoxic substance, or have the ability to bind to a cytotoxicsubstance, as described later. Furthermore, antigen-binding moleculeshaving a neutralizing activity instead of the properties of having acytotoxic activity, binding to a cytotoxic substance, or having theability to bind to a cytotoxic substance; or in addition to theseproperties are also suitable examples of a non-limiting embodiment.

Epitopes

“Epitope” means an antigenic determinant in an antigen, and refers to anantigen site to which the antigen-binding domain of an antigen-bindingmolecule disclosed herein binds. Thus, for example, the epitope can bedefined according to its structure. Alternatively, the epitope may bedefined according to the antigen-binding activity of an antigen-bindingmolecule that recognizes the epitope. When the antigen is a peptide orpolypeptide, the epitope can be specified by the amino acid residuesforming the epitope. Alternatively, when the epitope is a sugar chain,the epitope can be specified by its specific sugar chain structure.

A linear epitope is an epitope that contains an epitope whose primaryamino acid sequence has been recognized. Such a linear epitope typicallycontains at least three and most commonly at least five, for example,about 8 to about 10 or 6 to 20 amino acids in a specific sequence.

In contrast to the linear epitope, a “conformational epitope” is anepitope in which the primary amino acid sequence containing the epitopeis not the only determinant of the recognized epitope (for example, theprimary amino acid sequence of a conformational epitope is notnecessarily recognized by an epitope-defining antibody). Conformationalepitopes may contain a greater number of amino acids compared to linearepitopes. A conformational epitope-recognizing antibody recognizes thethree-dimensional structure of a peptide or protein. For example, when aprotein molecule folds and forms a three-dimensional structure, aminoacids and/or polypeptide main chains that form a conformational epitopebecome aligned, and the epitope is made recognizable by the antibody.Methods for determining epitope conformations include, for example, Xray crystallography, two-dimensional nuclear magnetic resonance,site-specific spin labeling, and electron paramagnetic resonance, butare not limited thereto. See, for example, Epitope Mapping Protocols inMethods in Molecular Biology (1996), Vol. 66, Morris (ed.).

The structure of the antigen-binding domain which binds to an epitope iscalled a paratope. An epitope and a paratope bind with stability throughthe action of hydrogen bonds, electrostatic force, van der Waals force,hydrophobic bonds, and such between the epitope and the paratope. Thisstrength of binding between the epitope and paratope is called affinity.The total sum of binding strength when a plurality of antigens and aplurality of antigen-binding molecules bind is referred to as avidity.When an antibody comprising a plurality of antigen-binding domains(i.e., multivalent antibody) or such binds to a plurality of epitopes,the affinity acts synergistically, and therefore avidity becomes higherthan affinity.

Binding Activity

Examples of a method for assessing the epitope binding by a testantigen-binding molecule containing an IL-6R antigen-binding domain aredescribed below. According to the examples below, methods for assessingthe epitope binding by a test antigen-binding molecule containing anantigen-binding domain for an antigen other than IL-6R, can also beappropriately conducted.

For example, whether a test antigen-binding molecule containing an IL-6Rantigen-binding domain recognizes a linear epitope in the IL-6R moleculecan be confirmed for example as mentioned below. A linear peptidecomprising an amino acid sequence forming the extracellular domain ofIL-6R is synthesized for the above purpose. The peptide can besynthesized chemically, or obtained by genetic engineering techniquesusing a region encoding the amino acid sequence corresponding to theextracellular domain in an IL-6R cDNA. Then, a test antigen-bindingmolecule containing an IL-6R antigen-binding domain is assessed for itsbinding activity towards a linear peptide comprising the amino acidsequence forming the extracellular domain. For example, an immobilizedlinear peptide can be used as an antigen by ELISA to evaluate thebinding activity of the antigen-binding molecule towards the peptide.Alternatively, the binding activity towards a linear peptide can beassessed based on the level that the linear peptide inhibits the bindingof the antigen-binding molecule to IL-6R-expressing cells. These testscan demonstrate the binding activity of the antigen-binding moleculetowards the linear peptide.

Whether a test antigen-binding molecule containing an IL-6Rantigen-binding domain recognizes a conformational epitope can beassessed as follows. IL-6R-expressing cells are prepared for the abovepurpose. A test antigen-binding molecule containing an IL-6Rantigen-binding domain can be determined to recognize a conformationalepitope when it strongly binds to IL-6R-expressing cells upon contact,but does not substantially bind to an immobilized linear peptidecomprising an amino acid sequence forming the extracellular domain ofIL-6R. Herein, “not substantially bind” means that the binding activityis 80% or less, generally 50% or less, preferably 30% or less, andparticularly preferably 15% or less compared to the binding activitytowards cells expressing human IL-6R.

Methods for assaying the binding activity of a test antigen-bindingmolecule containing an IL-6R antigen-binding domain towardsIL-6R-expressing cells include, for example, the methods described inAntibodies: A Laboratory Manual (Ed Harlow, David Lane, Cold SpringHarbor Laboratory (1988) 359-420). Specifically, the assessment can beperformed based on the principle of ELISA or fluorescence activated cellsorting (FACS) using IL-6R-expressing cells as antigen.

In the ELISA format, the binding activity of a test antigen-bindingmolecule containing an IL-6R antigen-binding domain towardsIL-6R-expressing cells can be assessed quantitatively by comparing thelevels of signal generated by enzymatic reaction. Specifically, a testpolypeptide complex is added to an ELISA plate onto whichIL-6R-expressing cells are immobilized. Then, the test antigen-bindingmolecule bound to the cells is detected using an enzyme-labeled antibodythat recognizes the test antigen-binding molecule. Alternatively, whenFACS is used, a dilution series of a test antigen-binding molecule isprepared, and the antibody binding titer for IL-6R-expressing cells canbe determined to compare the binding activity of the testantigen-binding molecule towards IL-6R-expressing cells.

The binding of a test antigen-binding molecule towards an antigenexpressed on the surface of cells suspended in buffer or the like can bedetected using a flow cytometer. Known flow cytometers include, forexample, the following devices:

FACSCanto™ II FACSAria™ FACSArray™ FACSVantage™ SE

FACSCalibur™ (all are trade names of BD Biosciences)

EPICS ALTRA HyPerSort Cytomics FC 500 EPICS XL-MCL ADC EPICS XL ADC

Cell Lab Quanta/Cell Lab Quanta SC (all are trade names of BeckmanCoulter).

Preferable methods for assaying the binding activity of a testantigen-binding molecule containing an IL-6R antigen-binding domaintowards an antigen include, for example, the following method. First,IL-6R-expressing cells are reacted with a test antigen-binding molecule,and then this is stained with an FITC-labeled secondary antibody thatrecognizes the antigen-binding molecule. The test antigen-bindingmolecule is appropriately diluted with a suitable buffer to prepare themolecule at a desired concentration. For example, the molecule can beused at a concentration within the range of 10 μg/ml to 10 ng/ml. Then,the fluorescence intensity and cell count are determined usingFACSCalibur (BD). The fluorescence intensity obtained by analysis usingthe CELL QUEST Software (BD), i.e., the Geometric Mean value, reflectsthe quantity of antibody bound to cells. That is, the binding activityof a test antigen-binding molecule, which is represented by the quantityof the test antigen-binding molecule bound, can be determined bymeasuring the Geometric Mean value.

Whether a test antigen-binding molecule containing an IL-6Rantigen-binding domain shares a common epitope with anotherantigen-binding molecule can be assessed based on the competitionbetween the two molecules for the same epitope. The competition betweenantigen-binding molecules can be detected by cross-blocking assay or thelike. For example, the competitive ELISA assay is a preferredcross-blocking assay.

Specifically, in cross-blocking assay, the IL-6R protein immobilized tothe wells of a microtiter plate is pre-incubated in the presence orabsence of a candidate competitor antigen-binding molecule, and then atest antigen-binding molecule is added thereto. The quantity of testantigen-binding molecule bound to the IL-6R protein in the wells isindirectly correlated with the binding ability of a candidate competitorantigen-binding molecule that competes for the binding to the sameepitope. That is, the greater the affinity of the competitorantigen-binding molecule for the same epitope, the lower the bindingactivity of the test antigen-binding molecule towards the IL-6Rprotein-coated wells.

The quantity of the test antigen-binding molecule bound to the wells viathe IL-6R protein can be readily determined by labeling theantigen-binding molecule in advance. For example, a biotin-labeledantigen-binding molecule is measured using an avidin/peroxidaseconjugate and appropriate substrate. In particular, cross-blocking assaythat uses enzyme labels such as peroxidase is called “competitive ELISAassay”. The antigen-binding molecule can also be labeled with otherlabeling substances that enable detection or measurement. Specifically,radiolabels, fluorescent labels, and such are known.

When the candidate competitor antigen-binding molecule can block thebinding by a test antigen-binding molecule containing an IL-6Rantigen-binding domain by at least 20%, preferably at least 20 to 50%,and more preferably at least 50% compared to the binding activity in acontrol experiment conducted in the absence of the competitorantigen-binding molecule, the test antigen-binding molecule isdetermined to substantially bind to the same epitope bound by thecompetitor antigen-binding molecule, or compete for the binding to thesame epitope.

When the structure of an epitope bound by a test antigen-bindingmolecule containing an IL-6R antigen-binding domain has already beenidentified, whether the test and control antigen-binding molecules sharea common epitope can be assessed by comparing the binding activities ofthe two antigen-binding molecules towards a peptide prepared byintroducing amino acid mutations into the peptide forming the epitope.

To measure the above binding activities, for example, the bindingactivities of test and control antigen-binding molecules towards alinear peptide into which a mutation is introduced are compared in theabove ELISA format. Besides the ELISA methods, the binding activitytowards the mutant peptide bound to a column can be determined byflowing test and control antigen-binding molecules in the column, andthen quantifying the antigen-binding molecule eluted in the elutionsolution. Methods for adsorbing a mutant peptide to a column, forexample, in the form of a GST fusion peptide, are known.

Alternatively, when the identified epitope is a conformational epitope,whether test and control antigen-binding molecules share a commonepitope can be assessed by the following method. First, IL-6R-expressingcells and cells expressing IL-6R with a mutation introduced into theepitope are prepared. The test and control antigen-binding molecules areadded to a cell suspension prepared by suspending these cells in anappropriate buffer such as PBS. Then, the cell suspensions areappropriately washed with a buffer, and an FITC-labeled antibody thatrecognizes the test and control antigen-binding molecules is addedthereto. The fluorescence intensity and number of cells stained with thelabeled antibody are determined using FACSCalibur (BD). The test andcontrol antigen-binding molecules are appropriately diluted using asuitable buffer, and used at desired concentrations. For example, theymay be used at a concentration within the range of 10 μg/ml to 10 ng/ml.The fluorescence intensity determined by analysis using the CELL QUESTSoftware (BD), i.e., the Geometric Mean value, reflects the quantity oflabeled antibody bound to cells. That is, the binding activities of thetest and control antigen-binding molecules, which are represented by thequantity of labeled antibody bound, can be determined by measuring theGeometric Mean value.

In the above method, whether an antigen-binding molecule does “notsubstantially bind to cells expressing mutant IL-6R” can be assessed,for example, by the following method. First, the test and controlantigen-binding molecules bound to cells expressing mutant IL-6R arestained with a labeled antibody. Then, the fluorescence intensity of thecells is determined. When FACSCalibur is used for fluorescence detectionby flow cytometry, the determined fluorescence intensity can be analyzedusing the CELL QUEST Software. From the Geometric Mean values in thepresence and absence of the polypeptide complex, the comparison value(ΔGeo-Mean) can be calculated according to Formula 1 below to determinethe ratio of increase in fluorescence intensity as a result of thebinding by the antigen-binding molecule.

ΔGeo-Mean=Geo-Mean (in the presence of the polypeptide complex)/Geo-Mean(in the absence of the polypeptide complex)  Formula 1

The Geometric Mean comparison value (ΔGeo-Mean value for the mutantIL-6R molecule) determined by the above analysis, which reflects thequantity of a test antigen-binding molecule bound to cells expressingmutant IL-6R, is compared to the ΔGeo-Mean comparison value thatreflects the quantity of the test antigen-binding molecule bound toIL-6R-expressing cells. In this case, the concentrations of the testantigen-binding molecule used to determine the ΔGeo-Mean comparisonvalues for IL-6R-expressing cells and cells expressing mutant IL-6R areparticularly preferably adjusted to be equal or substantially equal. Anantigen-binding molecule that has been confirmed to recognize an epitopein IL-6R is used as a control antigen-binding molecule.

If the ΔGeo-Mean comparison value of a test antigen-binding molecule forcells expressing mutant IL-6R is smaller than the ΔGeo-Mean comparisonvalue of the test antigen-binding molecule for IL-6R-expressing cells byat least 80%, preferably 50%, more preferably 30%, and particularlypreferably 15%, then the test antigen-binding molecule “does notsubstantially bind to cells expressing mutant IL-6R”. The formula fordetermining the Geo-Mean (Geometric Mean) value is described in the CELLQUEST Software User's Guide (BD biosciences). When the comparison showsthat the comparison values are substantially equivalent, the epitope forthe test and control antigen-binding molecules can be determined to bethe same.

Target Tissue

The term “target tissue” as used herein refers to a tissue containingcells carrying antigens to which the antigen-binding molecules of thepresent invention bind depending on the concentration of small moleculecompounds. It is a tissue that yields positive pharmacological effectsfor the organism carrying the tissue, when the antigen-binding moleculesbind to a membrane-type molecule expressed on the cells or bind to asoluble molecule present in the tissue. In this case, the phrase“positive pharmacological effects” refers to effects that relieve,alleviate, ameliorate, or cure symptoms brought about by pathologicalsites containing the target tissue for the organism carrying the tissue.When the symptoms are brought about by malignant tumors such as cancer,a non-limiting embodiment of a mechanism that yields such apharmacological effect is, for example, cytotoxic activity and growthinhibition against cancer cells, and immunostimulation in cancertissues. In the case of inflammatory diseases, examples of such anon-limiting embodiment of the mechanism include immunosuppression andactivity to block actions of inflammatory cytokines in inflammatorytissues.

Cancer Tissue-Specific Compounds

The term “compound specific to a cancer tissue (cancer tissue-specificcompound)” as used herein refers to a compound differentially present incancer tissues as compared to non-cancerous tissues. Herein, the term“cancer” is generally used to describe malignant neoplasms, which may bemetastatic or non-metastatic. Non-limiting examples of carcinomasdeveloped from epithelial tissues such as skin or digestive tractinclude brain tumor, skin cancer, head and neck cancer, esophagealcancer, lung cancer, stomach cancer, duodenal cancer, breast cancer,prostate cancer, cervical cancer, endometrial cancer, pancreatic cancer,liver cancer, colorectal cancer, colon cancer, bladder cancer, andovarian cancer. Non-limiting examples of sarcomas developed fromnon-epithelial (interstitial) tissues such as muscles includeosteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma,liposarcoma, and angiosarcoma. Non-limiting examples of hematologicalcancer derived from hematopoietic organs include malignant lymphomasincluding Hodgkin's lymphoma and non Hodgkin's lymphoma; leukemiaincluding acute myelocytic leukemia or chronic myelocytic leukemia, andacute lymphatic leukemia or chronic lymphatic leukemia; and multiplemyeloma. The term “neoplasm” widely used herein refers to any newlyformed diseased tissue tumor. In the present invention, neoplasms causeformation of tumors, which are partly characterized by angiogenesis.Neoplasms may be benign such as hemangioma, glioma, or teratoma, ormalignant such as carcinoma, sarcoma, glioma, astrocytoma,neuroblastoma, or retinoblastoma.

The term “cancer tissue” refers to a tissue containing at least onecancer cell. Therefore, as cancer tissues contain cancer cells and bloodvessels, it refers to all cell types contributing to the formation of atumor mass containing cancer cells and endothelial cells. Herein, “tumormass” refers to a foci of tumor tissue. The term “tumor” is generallyused to mean a benign neoplasm or a malignant neoplasm.

For example, in several embodiments, cancer tissue-specific compoundsmay be compounds defined by qualitative properties of cancer tissuessuch as being present in cancer tissues but absent in non-cancertissues, or being absent in cancer tissues but present in non-cancertissues. In other embodiments, cancer tissue-specific compounds may becompounds defined by quantitative properties of cancer tissues such asbeing present in cancer tissues at a concentration different (forexample, higher concentration or lower concentration) from that innon-cancer tissues. For example, cancer tissue-specific compounds arepresent differentially at arbitrary concentrations. Generally, cancertissue-specific compounds can be present at a concentration increased byat least 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 100%, atleast 110%, at least 120%, at least 130%, at least 140%, at least 150%,at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, atleast 100-fold, at least 10³-fold, at least 10⁴-fold, at least 10⁵-fold,at least 10⁶-fold, or more, or up to infinity (i.e., when the compoundis absent in non-cancerous tissues). Alternatively, they can generallybe present at a concentration decreased by at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 100% (i.e., absent). Preferably, cancertissue-specific compounds are differentially present at statisticallysignificant concentrations (that is, as determined using either Welch'st-test or Wilcoxon rank sum test, the p value is less than 0.05 and/orthe q value is less than 0.10). Examples of a non-limiting embodiment ofa cancer tissue-specific compound include compounds which are cancertissue-specific metabolites produced by metabolic activitiescharacteristic of cancer cells, immune cells, or stromal cells containedin cancer tissues, such as those described below (cancer tissue-specificmetabolites, cancer cell-specific metabolites, metabolites specific toimmune cells that infiltrated into cancer tissues, and cancer stromalcell-specific metabolites).

The term “unnatural compound” as used herein refers to an unnaturallyderived chemical substance and its metabolites. An embodiment of theinvention is an unnaturally derived chemical substance that has theproperty of accumulating at the target tissue after being administeredto a living body from outside the body, and metabolites thereof.Examples of an unnatural compound include (1) Capecitabine (Xeloda) andits metabolite 5-FU (fluorouracil), and (2) TH-302 andbromo-isophosphoramide mustard (Br-IPM). 5-FU is a metabolite ofCapecitabine (Xeloda), and is known to be metabolized by cytidinedeaminase and thymidine phosphorylase which are metabolic enzymesspecific in cancer tissues (Desmoulin F. et al. Drug Metab Dispos.2002). TH-302 is known to be converted to Br-IPM by reduction under alow-oxygen condition as in the periphery of cancer tissues (Duan J X, etal. J Med Chem. 2008). For example, when Capecitabine (Xeloda) isadministered, it is metabolized into 5-FU by cancer-specific metabolicenzymes, and therefore, the concentration of 5-FU becomes high at thecancer site (Desmoulin F. et al. Drug Metab Dispos. 2002). Accordingly,antibodies that use 5-FU as their switch may be able to bind selectivelyto the target antigen only at the cancer site. Furthermore, besidesmetabolic enzymes, molecules formed in a low-oxygen environment or anacidic environment specific to cancers may also be used as the switch.For example, TH-302 (Duan J X, et al. J Med Chem. 2008) is metabolizedinto Br-IPM under a low-oxygen condition, and therefore, antibodies thatuse Br-IPM as their switch may be able to bind selectively to the targetantigen only at the cancer site. Examples of administration methods ofthe unnatural compound to a living body include known administrationmethods such as oral administration, administration throughinstillation, transdermal administration, transnasal administration,intravenous administration, and transpulmonary administration, but arenot limited thereto.

Besides chemical substances that have the property of accumulating atthe target tissue and metabolites thereof, another embodiment of theterm “unnatural compound” used herein also includes chemical substancesand such, which is an unnatural compound that serves as a switch thatcan control the action of antibodies through intake of oral agents, forexample, by oral administration. More specifically, they are chemicalsubstances and such, which is an unnatural compound that can beadministered non-invasively such as orally and serves as a switch thatcan control the antibody action, when a switch antibody that binds to acertain antigen is initially administered invasively such asintravenously or subcutaneously; and then an exogenous compound thatserves as a switch is administered non-invasively such as orally.Examples of such compounds include ATPγS and kynurenine metabolites, butare not limited thereto. The problem with antibody pharmaceuticals isthat since they have a long half-life, side-effects are lasting whenthese effects occur; however, if effects of such antibodies can becontrolled by non-invasive administration such as oral administration ofan unnatural compound, effects of the pharmaceuticals can be stopped byterminating administration of the switch molecule when side effectsoccur. Furthermore, by preliminary administration of a switch antibody,administration of the switch molecule only when symptoms due to thedisorder occur, and pharmacological effects exertion by non-invasiveadministration such as oral administration only when necessary, areallowed.

The “antigen-binding molecules comprising an antigen-binding domainwhose antigen-binding activity varies depending on the concentration ofan unnatural compound” of the present invention may yield positivepharmacological effects when administered to a living body.

Cancer Tissue-Specific Metabolites

The term “metabolism” refers to chemical changes that take place inbiological tissues and includes “anabolism” and “catabolism”. Anabolismrefers to biosynthesis or accumulation of molecules, and catabolismrefers to degradation of molecules. “Metabolites” are intermediates orproducts that arise from metabolism. “Primary metabolites” refers tometabolites directly involved in the process of growth or proliferationof cells or organisms. “Secondary metabolites” refer to products thatare not directly involved in such process of growth or proliferation,and are products such as pigments or antibiotics that are produced as aresult of metabolism which biosynthesizes substances that are notdirectly involved in biological phenomena common to cells and organisms.The metabolites may be metabolites of “biopolymers”, or they may bemetabolites of “small molecules”. “Biopolymers” are polymers comprisingone or more types of repeating units. Biopolymers are generally found inbiological systems, and examples include cells forming the organism andintercellular matrices that adhere to them, molecules having a molecularweight of approximately 5000 or more which form structures such asinterstitial matrices, particularly polysaccharides (carbohydrates andsuch), peptides (this term is used so as to include polypeptides andproteins), and polynucleotides, and similarly their analogs such ascompounds composed of or including amino acid analogs or non-amino acidgroups.

As used herein, the term “small molecules” refers to natural chemicalsubstances other than “biopolymers” that exist in vivo or unnaturalchemical substances, and are preferably target tissue-specific compoundsor unnatural compounds but are not limited thereto. Suitable examples ofa non-limiting embodiment of a cancer tissue-specific metabolitedescribed herein include cancer cell-specific small-molecule metabolites(Eva Gottfried, Katrin Peter and Marina P. Kreutz, From Molecular toModular Tumor Therapy (2010) 3 (2), 111-132). In addition, metabolitesthat are highly produced by immune cells that infiltrate into cancertissues, and metabolites that are highly produced by stromal cells thatsupport the survival and/or growth of cancer cells (cancer stromal cellsor cancer associated stromal fibroblasts (CAF)) are also included.Infiltrating immune cells are, for example, dendritic cells, inhibitorydendritic cells, inhibitory T cells, exhausted T cells, and myelomaderived suppressor cells (MDSC). Furthermore, metabolites of the presentinvention include compounds released from inside the cells to outsidethe cells when cells present in cancer tissues (cancer cells, immunecells, or stromal cells) die due to apoptosis, necrosis, or such.

To identify cancer cell-specific metabolites, metabolomic analysesfocused on metabolic profiling can be suitably used, in addition totranscriptome-level analyses (for example, Dhanasekaran et al. (Nature(2001) 412, 822-826), Lapointe et al. (Proc. Natl. Acad. Sci. U.S.A.(2004) 101, 811-816) or Perou et al. (Nature (2000) 406, 747-752)) andproteome-level analyses (for example, Ahram et al. (Mol. Carcinog.(2002) 33, 9-15), Hood et al. (Mol. Cell. Proteomics (2005) 4,1741-1753)). More specifically, to identify metabolites in test samples,metabolic profiling that uses high-pressure liquid chromatography(HPLC), nuclear magnetic resonance (NMR) (Brindle et al. (J. Mol.Recognit. (1997) 10, 182-187), mass spectrometry (Gates and Sweeley(Clin. Chem. (1978) 24, 1663-1673) (GC/MS and LC/MS)), and ELISA or suchindividually and/or in combination may be used appropriately.

These studies elucidated heterogeneity within the constituted tumorswhich results from changing the concentration gradient of growth factorsand metabolites (glucose, oxygen, or such) that enable cancer cellgrowth under low oxygen pressure conditions (Dang and Semenza (TrendsBiochem. Sci. (1999) 24, 68-72)). In these studies, cell line models arealso used to understand the change in energy utilization pathwaydepending on the different malignancy levels of tumors (Vizan et al.(Cancer Res. (2005) 65, 5512-5515)). Examples of a non-limitingembodiment of the technical components of the metabolomics platforminclude sample extraction, separation, detection, spectroscopicanalysis, data normalization, description of class-specific metabolites,pathway mapping, confirmation, and functional characterization ofcandidate metabolites described by Lawton et al. (Pharmacogenomics(2008) 9, 383). These methods enable identification of cancercell-specific metabolites in desired cancer tissues.

Examples of a non-limiting embodiment of cancer tissue-specificcompounds or cancer tissue-specific metabolites used in the presentinvention preferably include at least one compound selected from thecompounds below. At least one compound means that in addition to caseswhere the antigen-binding activity of a same antigen-binding domaindescribed below depends on one type of cancer tissue-specific compoundor metabolite, cases where it depends on several types of cancertissue-specific compounds or metabolites are included.

(1) Primary Metabolites of the Krebs Cycle or of the Glycolytic SystemSuch as Lactic Acid, Succinic Acid, and Citric Acid

Preferable examples of a non-limiting embodiment of a cancertissue-specific compound, particularly a cancer cell-specificmetabolite, used in the present invention include primary metabolitessuch as lactic acid, succinic acid, and citric acid, which are producedas a result of glucose metabolism, and are present at higherconcentrations in cancer tissues as compared to in the surroundingnon-cancerous tissues. The glycolytic system phenotype, which ischaracterized as an up-regulation of enzymes of the glycolytic system(Embden-Meyerhof pathway) such as pyruvate kinase, hexokinase, andlactic acid dehydrogenase (LDH), has been conventionally known to be acharacteristic of solid tumors as Warburg effect.

That is, in tumor cells, high expression of the pyruvate kinase isoformM2 which is necessary for anaerobic glycolysis, and not isoform M1, isconsidered to be working advantageously for the growth of tumor cells invivo (Christofk et al. (Nature (2008) 452, 230-233). Pyruvic acidproduced by pyruvate kinase is subjected to feedback inhibition bylactic acid produced as a result of equilibrium reaction by lactic aciddehydrogenase (LDH) under anaerobic conditions. Since the feedbackinhibition causes promotion of respiration in mitochondria (Krebs cycle)and cell growth inhibition, up regulation of LDH, hexokinase, andglucose transporter (GLUT) is said to play an important role in theproliferation of cancer cells (Fantin et al. (Cancer Cell (2006) 9,425-434)). Glucose is metabolized by the glycolytic system, and thefinal metabolite lactic acid is transported together with protons to thetumor surrounding, and as a result, the pH of the tissues surroundingthe tumor is said to become acidic. Lactic acid, which is the finalproduct of the glycolytic pathway, as well as succinic acid and citricacid produced by promotion of respiration in mitochondria are known tobe accumulated in cancer tissues (Teresa et al. (Mol. Cancer (2009) 8,41-59)). Examples of a non-limiting embodiment of cancer tissue-specificcompounds, particularly cancer cell-specific metabolites, used in thepresent invention preferably include such primary metabolites such aslactic acid, succinic acid, and citric acid produced by metabolism bythe glycolytic pathway. Furthermore, succinic acid which is present athigh concentration in cells is known to leak out to the outside of thecells upon cell death (Nature Immunology, (2008) 9, 1261-1269).Therefore, succinic acid concentration is thought to be increased incancer tissues in which cell death occurs frequently.

(2) Amino Acids Such as Alanine, Glutamic Acid, and Aspartic Acid

Besides the above-mentioned glucose metabolism, the amino acidmetabolism is also known to be altered in tumor cells which requirecontinuous supply of essential amino acids and non-essential amino acidsthat are necessary for the biosynthesis of biopolymers under anaerobicconditions. Glutamine which contains two nitrogens in its side chainacts as a nitrogen transporter, and is an amino acid that is most widelydistributed in an organism. Tumor cells, in which the rate of glutamineuptake into cells is increased, is said to be functioning as a glutaminetrap. Such increase in the uptake of glutamine and activity ofconverting into glutamic acid and lactic acid is called“glutaminolysis”, and is considered to be a characteristic oftransformed (tumor) cells (Mazurek and Eigenbrodt (Anticancer Res.(2003) 23, 1149-1154); and Mazurek et al. (J. Cell. Physiol. (1999) 181,136-146)). As a result, cancer patients show an increase in glutamicacid concentration while showing a decrease in plasma glutamine level(Droge et al. (Immunobiology (1987) 174, 473-479)). Furthermore,correlation was observed between concentrations of ¹³C-labeled succinicacid, ¹³C-labeled alanine, ¹³C-labeled glutamic acid, and ¹³C-labeledcitric acid in studies on ¹³C-radiolabeled glucose metabolism in lungcancer tissues. Suitable examples of a non-limiting embodiment of cancertissue-specific compounds used in this invention include alanine,glutamic acid, and aspartic acid which accumulate at high concentrationsin cancer tissues through such glutaminolysis and the like.

(3) Amino Acid Metabolite Such as Kynurenine

Indolamine 2,3-dioxygenase (IDO) is a tryptophan-metabolizing enzymewhich is highly expressed in many cancers such as melanoma, coloncancer, and kidney cancer (Uyttenhove et al. (Nat. Med. (2003) 9,1269-127)); and it is known to have two isoforms (Lob et al. (CancerImmunol. Immunother. (2009) 58, 153-157)). IDO catalyzes the conversionof tryptophan to kynurenine (shown as Compound 1), and is the firstenzyme in the nicotinamide nucleotide (NAD) de novo pathway.Furthermore, in glioma which does not express IDO, kynurenine isproduced from tryptophan by tryptophan 2,3-dioxygenase (TDO) in theliver (Opitz et al. (Nature (2011) 478, 7368, 197-203)). IDO is alsoexpressed in dendritic cells infiltrated into cancer tissues, anddendritic cells also produce kynurenine (J. Immunol. (2008) 181,5396-5404). IDO is also expressed in myeloid-derived suppressor cells(MDSC) in cancer tissues, and MDSC also produces kynurenine (Yu et al.(J. Immunol. (2013) 190, 3783-3797)).

Kynurenine is known to suppress the same type of T cell response(Frumento et al. (J. Exp. Med. (2002) 196, 459-468); and a mechanism hasbeen suggested, in which tumor cells evade antitumor immune responsesthrough such inhibition, and proliferation of glioma cells is promotedthrough an autocrine proliferation mechanism in which kynurenine acts asan endogenous ligand for the aryl hydrocarbon receptor expressed ongliomas (Optiz et al. (mentioned above)). Kynurenine is converted toanthranilic acid (shown as Compound 2) by kynurenidase, and to3-hydroxykynurenine (shown as Compound 3) by kynurenine 3-hydroxylase.Anthranilic acid and 3-hydroxykynurenine are both converted to3-hydroxyanthranilic acid, the precursor of NAD.

Kynurenine is converted to kynurenic acid (shown as Compound 4) bykynurenine aminotransferase. Examples of a non-limiting embodiment ofcancer tissue-specific compounds, particularly cancer cell-specificmetabolites, used in the present invention preferably include such aminoacid metabolites such as kynurenine and its metabolites such asanthranilic acid, 3-hydroxykynurenine, and kynurenic acid.

(4) Arachidonic Acid Metabolites Such as Prostaglandin E2

Prostaglandin E2 (PGE2) (Compound 5) is an arachidonic acid metabolitecalled a prostanoid, which includes thromboxane and prostaglandinsynthesized by cyclooxygenase (COX)-1/2 (Warner and Mitchell (FASEB J.(2004) 18, 790-804)). PGE2 promotes the proliferation of colon cancercells and suppresses their apoptosis (Sheng et al. (Cancer Res. (1998)58, 362-366)). Cyclooxygenase expression is known to be altered in manycancer cells. More specifically, while COX-1 is expressed constitutivelyin almost all tissues, COX-2 has been found to be mainly induced bycertain types of inflammatory cytokines and cancer genes in tumors(Warner and Mitchell (mentioned above)). In addition, COX-2overexpression has been reported to be related to bad prognosis forbreast cancer (Denkert et al. (Clin. Breast Cancer (2004) 4, 428-433)),and rapid disease progression for ovarian cancer (Denker et al. (Mod.Pathol. (2006) 19, 1261-1269)). Inhibitory T cells that have infiltratedinto cancer tissues also produce prostaglandin E2 (Curr. Med. Chem.(2011) 18, 5217-5223). Small molecules such as the arachidonic acidmetabolites prostaglandin and leukotriene are known to act as astimulating factor that regulates autocrine and/or paracrine growth ofcancer (Nat. Rev. Cancer (2012) 12 (11) 782-792). Examples of anon-limiting embodiment of cancer tissue-specific compounds used in thepresent invention, particularly cancer cell-specific metabolites andimmune cell-specific metabolites that have infiltrated into cancertissues, preferably include such arachidonic acid metabolites such asprostaglandin E2. Besides prostaglandin E2, production of thromboxane A2(TXA2) is enhanced in cancer tissues such as colorectal cancer tissues(J. Lab. Clin. Med. (1993) 122, 518-523), and thromboxane A2 can besuitably presented as a non-limiting embodiment of an arachidonic acidmetabolite of the present invention.

(5) Nucleosides Carrying a Purine Ring Structure Such as Adenosine,Adenosine Triphosphate (ATP), Adenosine Diphosphate (ADP), and AdenosineMonophosphate (AMP)

When cancer cells undergo cell death, a large amount of ATP in the cellis known to leak out to the outside of the cells. Therefore, the ATPconcentration is remarkably higher in cancer tissues than in normaltissues (PLoS One. (2008) 3, e2599). Multiple types of cells releaseadenine nucleotides in the form of ATP, ADP, and AMP. Metabolism takesplace through an extracellular enzyme on the cell surface such asextracellular 5′-nucleotidase (ecto-5′-nucleotidase) (CD73) (Resta andThompson (Immunol. Rev. (1998) 161, 95-109) and Sadej et al. (MelanomaRes. (2006) 16, 213-222)). Adenosine is a purine nucleoside that existsconstitutively at low concentration in the extracellular environment,but in hypoxic tissues found in solid cancers, a remarkable increase inthe extracellular adenosine concentration has been reported (Blay andHoskin (Cancer Res. (1997) 57, 2602-2605). CD73 is expressed on thesurface of immune cells and tumors (Kobie et al. (J. Immunol. (2006)177, 6780-6786)), and its activity has been found to be increased inbreast cancer (Canbolat et al. (Breast Cancer Res. Treat. (1996) 37,189-193)), stomach cancer (Durak et al. (Cancer Lett. (1994) 84,199-202)), pancreatic cancer (Flocke and Mannherz (Biochim. Biophys.Acta (1991) 1076, 273-281), and glioblastoma (Bardot et al. (Br. J.Cancer (1994) 70, 212-218)). It has been proposed that the accumulationof adenosine in cancer tissues may be caused by an increase in theintracellular adenosine production through dephosphorylation of AMP by5′-nucleotidase in the cytoplasm (Headrick and Willis (Biochem. J.(1989) 261, 541-550)). Furthermore, inhibitory T cells and such thathave infiltrated into cancer tissues also express ATPase and produceadenosine (Proc. Natl. Acad. Sci. (2006) 103 (35), 13132-13137; Curr.Med. Chem. (2011) 18, 5217-5223). The produced adenosine is consideredto be rendering the cancer tissue an immunosuppressive environmentthrough adenosine receptors such as the A2A receptor (Curr. Med. Chem.(2011), 18, 5217-23). Examples of a non-limiting embodiment of thecancer tissue-specific compound used in the present invention preferablyinclude ATP, ADP, AMP, and adenosine which accumulate at highconcentration in cancer tissues through such metabolism of purinenucleotides such as ATP. Furthermore, since adenosine is degraded toinosine by adenosine deaminase, inosine accumulates at highconcentration.

(6) Uric Acid

Uric acid is a product of the metabolic pathway of purine nucleosides invivo, and is released to the outside of cells such as the interstitialspace and blood. In recent years, it has been found to be released fromdead cells that are present at sites of lesions such as cancer tissues(Nat. Med. (2007) 13, 851-856). Examples of a non-limiting embodiment ofcancer tissue-specific compounds used in the present inventionpreferably include such uric acid which accumulates at highconcentration in cancer tissues due to metabolism of purine nucleotidessuch as ATP.

(7) 1-Methyl nicotinamide

The enzyme nicotinamide N-methyl transferase is known to be highlyexpressed in several human cancer tissues. When this enzyme produces thestable metabolite 1-methylnicotinamide from nicotinamide, the methylgroup of S-adenosylmethionine (SAM) which serves as a methyl donor isconsumed; therefore, the high expression of nicotinamideN-methyltransferase has been suggested to contribute to tumorigenesisthrough a mechanism that impairs the DNA methylation abilityaccompanying a decrease in the SAM concentration in cancer cells(Ulanovskaya et al. (Nat. Chem. Biol. (2013) 9 (5) 300-306)). The stablemetabolite of this enzyme, 1-methylnicotinamide is known to be secretedto the outside of cancer cells (Yamada et al. (J. Nutr. Sci. Vitaminol.(2010) 56, 83-86)), and preferable examples of a non-limiting embodimentof cancer tissue-specific compounds used in the present inventioninclude 1-methylnicotinamide and such which accumulate at highconcentration in cancer tissues through nicotinamide metabolism.

Inflammatory Tissue-Specific Compounds

The term “compound specific to inflammatory tissue (inflammatorytissue-specific compound)” as used herein refers to a compound that ispresent differentially in inflammatory tissues as compared tonon-inflammatory tissues. Herein, examples of “inflammatory tissues”include:

joints with rheumatoid arthritis or osteoarthritis;lungs (alveoli) with bronchial asthma or COPD;digestive organs of inflammatory bowel disease, Crohn's disease, orulcerative colitis;fibrotic tissues of fibrosis of the liver, kidney, or lung;tissues undergoing rejection reaction in organ transplantation;blood vessels and heart (myocardium) in arteriosclerosis or heartfailure;visceral fat in metabolic syndrome;skin tissues in atopic dermatitis or other dermatitis; andspinal nerves in disk herniation or chronic low back pain.

Inflammatory Tissue-Specific Metabolites

“Inflammatory tissue-specific metabolite” refers to metabolites highlyproduced by immune cells that have infiltrated into inflammatorytissues, and metabolites highly produced by specifically normal cellsthat have been damaged in inflammatory tissues. Examples of infiltratingimmune cells include effector T cells, mature dendritic cells,neutrophils, granule cells (mast cells), and basophils. Furthermore,metabolites in the present invention include compounds that are releasedfrom inside the cells to the outside of the cells when the cells thatare present in inflammatory tissues (immune cells and normal cells) dieby apoptosis, necrosis, or such.

Examples of a non-limiting embodiment of the inflammatorytissue-specific compounds or inflammatory tissue-specific metabolitesused in the present invention preferably include at least one compoundselected from the compounds below. At least one compound means includingcases where the antigen-binding activity of a same antigen-bindingdomain described below depends on one type of inflammatorytissue-specific compound or metabolite, as well as cases where itdepends on several types of inflammatory tissue-specific compounds ormetabolites.

(1) Arachidonic Acid Metabolites Such as Prostaglandin E2

The PGE2 concentration has been known to be high in rheumatoid arthritisand osteoarthritis (Eur. J. Clin. Pharmacol. (1994) 46, 3-7.; Clin. Exp.Rheumatol. (1999) 17, 151-160; Am. J. Vet. Res. (2004) 65, 1269-1275).Examples of a non-limiting embodiment of inflammatory tissue-specificcompounds, particularly inflammatory tissue-specific metabolites andmetabolites specific to immune cells that infiltrate into inflammatorytissues used in the present invention preferably include sucharachidonic acid metabolites such as prostaglandin E2.

(2) Nucleosides Carrying a Purine Ring Structure Such as Adenosine,Adenosine Triphosphate (ATP), Adenosine Diphosphate (ADP), and AdenosineMonophosphate (AMP)

ATP concentration is known to be high in pulmonary alveoli whereinflammation caused by bronchial asthma is taking place (Nat. Med.(2007) 13, 913-919). ATP concentration is also known to be high inpulmonary alveoli where inflammation caused by COPD is taking place (Am.J. Respir. Crit. Care Med. (2010) 181, 928-934). Furthermore, adenosineconcentration has been observed to be high in the joint fluid ofrheumatoid arthritis patients (Journal of Pharmaceutical and BiomedicalAnalysis (2004) 36, 877-882). Furthermore, ATP concentration is known tobe high in tissues where a rejection reaction is taking place due toGVHD (Nat. Med. (2010) 16, 1434-1438). Adenosine concentration is knownto be enhanced in fibrotic tissues of the liver, kidney, and lung (FASEBJ. (2008) 22, 2263-2272; J. Immunol. (2006) 176, 4449-4458; J. Am. Soc.Nephrol. (2011) 22 (5), 890-901; PLoS ONE J. (2010) 5 (2), e9242).Furthermore, ATP concentration has been observed to be increased infibrotic tissues of pulmonary fibrosis patients (Am. J. Respir. Crit.Care Med. (2010) 182, 774-783). Examples of a non-limiting embodiment ofan inflammatory tissue-specific compound used in the present inventionsuitably include ATP, ADP, AMP, adenosine and such which accumulate athigh concentration in inflammatory tissues by metabolism of such purinenucleotides such as ATP. In addition, inosine accumulates at a highconcentration due to degradation of adenosine by adenosine deaminase toproduce inosine.

(3) Uric Acid

Uric acid is a product of the metabolic pathway of purine nucleosides invivo, and is released to the outside of cells such as the interstitialspace and blood. In recent years, uric acid released from cellsundergoing necrosis has been found to promote inflammatory response (J.Clin. Invest. (2010) 120 (6), 1939-1949). Examples of a non-limitingembodiment of inflammatory tissue-specific compounds to be used in thepresent invention suitably include such uric acid which accumulates athigh concentration in inflammatory tissues due to metabolism of purinenucleotides such as ATP.

Antigen-Binding Domain

Herein, an “antigen-binding domain” may be of any structure as long asit binds to an antigen of interest. Such domains preferably include, forexample:

antibody heavy-chain and light-chain variable regions;a module of about 35 amino acids called A domain which is contained inthe in vivo cell membrane protein Avimer (International Publication No.WO 2004/044011, International Publication No. WO 2005/040229);Adnectin containing the 10Fn3 domain which binds to the protein moietyof fibronectin, a glycoprotein expressed on cell membrane (InternationalPublication No. WO 2002/032925);Affibody which is composed of a 58-amino acid three-helix bundle basedon the scaffold of the IgG-binding domain of Protein A (InternationalPublication No. WO 1995/001937);Designed Ankyrin Repeat proteins (DARPins) which are a region exposed onthe molecular surface of ankyrin repeats (AR) having a structure inwhich a subunit consisting of a turn comprising 33 amino acid residues,two antiparallel helices, and a loop is repeatedly stacked(International Publication No. WO 2002/020565);Anticalins and such, which are domains consisting of four loops thatsupport one side of a barrel structure composed of eight circularlyarranged antiparallel strands that are highly conserved among lipocalinmolecules such as neutrophil gelatinase-associated lipocalin (NGAL)(International Publication No. WO 2003/029462); andthe concave region formed by the parallel-sheet structure inside thehorseshoe-shaped structure constituted by stacked repeats of theleucine-rich-repeat (LRR) module of the variable lymphocyte receptor(VLR) which does not have the immunoglobulin structure and is used inthe system of acquired immunity in jawless vertebrate such as lampreyand hagfish (International Publication No. WO 2008/016854).

Suitable examples of the antigen-binding domains of the presentinvention include antigen-binding domains comprising antibodyheavy-chain and light-chain variable regions. Examples of suchantigen-binding domains are suitably “single chain Fv (scFv)”, “singlechain antibody”, “Fv”, “single chain Fv 2 (scFv2)”, “Fab”, or “F(ab′)2”.

The antigen-binding domains of antigen-binding molecules of the presentinvention can bind to an identical epitope. Such identical epitope canbe present, for example, in a protein comprising the amino acid sequenceof SEQ ID NO: 1. Alternatively, each of the antigen-binding domains ofantigen-binding molecules of the present invention can bind to adifferent epitope. Herein, the different epitope can be present in, forexample, a protein comprising the amino acid sequence of SEQ ID NO: 1.

Specificity

“Specific” means that one of the molecules that specifically bind doesnot substantially bind to molecules other than the single or pluralityof partner molecules it binds to. Furthermore, “specific” is also usedwhen an antigen-binding domain is specific to a particular epitope amongmultiple epitopes in an antigen. When an epitope bound by anantigen-binding domain is contained in multiple different antigens,antigen-binding molecules containing the antigen-binding domain can bindto various antigens that have the epitope. Here, “does not substantiallybind” is determined according to the method described in theabove-mentioned section on binding activity, and refers to the bindingactivity of a molecule that specifically binds to a molecule other thanthe partner molecule, where the binding activity is not more than 80%,normally not more than 50%, preferably not more than 30%, orparticularly preferably not more than 15% of the binding activity to itspartner molecule.

Cytotoxic Activity

In a non-limiting embodiment, the present invention providesantigen-binding molecules that comprise an antigen-binding domain whoseantigen-binding activity varies depending on the concentration of asmall molecule compound (e.g., cancer tissue-specific compound,inflammatory tissue-specific compound, or metabolites thereof) and whichhave cytotoxic activity against cells expressing a membrane-typemolecule on their cell membrane; and pharmaceutical compositionscomprising these antigen-binding molecules as an active ingredient. Inthe present invention, cytotoxic activity includes, for example,antibody-dependent cell-mediated cytotoxicity (ADCC) activity,complement-dependent cytotoxicity (CDC) activity, and cytotoxic activityby T cells. In the present invention, CDC activity refers to cytotoxicactivity by the complement system. On the other hand, ADCC activityrefers to the activity of immune cells to damage target cells when theimmune cells and such bind to the Fc region of antigen-binding moleculescomprising an antigen-binding domain that binds to a membrane-typemolecule expressed on the cell membrane of target cells via an Fcγreceptor expressed on the immune cells. Whether an antigen-bindingmolecule of interest has an ADCC activity or whether it has a CDCactivity can be determined using known methods (for example, CurrentProtocols in Immunology, Chapter 7. Immunologic studies in humans,Editor, Coligan et al., (1993)).

Specifically, effector cells, complement solution, and target cells arefirst prepared.

(1) Preparation of Effector Cells

Spleen is removed from a CBA/N mouse or the like, and spleen cells aredispersed in an RPMI1640 medium (Invitrogen). After the cells are washedin the same medium containing 10% fetal bovine serum (FBS, HyClone),effector cells are prepared by adjusting the spleen cell concentrationto 5×10⁶/mL.

(2) Preparation of Complement Solution

Baby Rabbit Complement (CEDARLANE) is diluted 10-fold in a culturemedium (Invitrogen) containing 10% FBS to prepare a complement solution.

(3) Preparation of Target Cells

The target cells can be radioactively labeled by culturing cellsexpressing the antigen with 0.2 mCi of ⁵¹Cr-sodium chromate- (GEHealthcare Bio-Sciences) in a DMEM medium containing 10% FBS for onehour at 37° C. After radioactive labeling, cells are washed three timesin an RPMI1640 medium containing 10% FBS, and the target cells can beprepared by adjusting the cell concentration to 2×10⁵/mL.

ADCC activity or CDC activity can be measured by the method describedbelow. In the case of ADCC activity measurement, 50 μL each of thetarget cell and antigen-binding molecule are added to a 96-well U-bottomplate (Becton Dickinson), and allowed to react for 15 minutes at roomtemperature. Then, 100 μL of effector cells are added to the plate andthis plate is placed in a carbon dioxide incubator for four hours. Thefinal concentration of the antigen-binding molecule may be set, forexample, to 0 μg/mL or 10 μg/mL. After incubation, 100 μL of thesupernatant is collected from each well, and the radioactivity ismeasured with a gamma counter (COBRAII AUTO-GAMMA, MODEL D5005, PackardInstrument Company). The cytotoxic activity (%) can be calculated usingthe measured values according to the equation: (A−C)/(B−C)×100. Arepresents the radioactivity (cpm) in each sample, B represents theradioactivity (cpm) in a sample to which 1% NP-40 (Nacalai Tesque) hasbeen added, and C represents the radioactivity (cpm) of a samplecontaining the target cells alone.

Meanwhile, in the case of CDC activity measurement, 504 of target celland 504 of an antigen-binding molecule are added to a 96-wellflat-bottomed plate (Becton Dickinson), and allowed to react for 15minutes on ice. Then, 100 μL of a complement solution is added to theplate, and this plate is placed in a carbon dioxide incubator for fourhours. The final concentration of the antigen-binding molecule may beset, for example, to 0 μg/mL or 3 μg/mL. After incubation, 100 μL ofsupernatant is collected from each well, and the radioactivity ismeasured with a gamma counter. The cytotoxic activity can be calculatedin the same way as in the determination of ADCC activity.

The later-described modified antigen-binding molecules to whichcytotoxic substances such as chemotherapeutic agents, toxic peptides, orradioactive chemical substances have been ligated can also be suitablyused as the antigen-binding molecules of the present invention havingcytotoxic activity. Such modified antigen-binding molecules (hereinafterreferred to as “antigen-binding molecule-drug conjugate”) can beobtained by chemically modifying the obtained antigen-binding molecules.Methods that have been already established in the field of antibody-drugconjugates and such may be used appropriately as a method for modifyingantigen-binding molecules. Furthermore, a modified antigen-bindingmolecule with a linked toxic peptide can be obtained by expressing in anappropriate host cell a fusion gene produced by linking a gene encodingthe toxic peptide in frame with a gene encoding an antigen-bindingmolecule of the present invention, and then isolating the molecule fromthe culture solution of the cells.

Neutralizing Activity

The present invention provides in a non-limiting embodiment apharmaceutical composition that induces an immune response, comprisingas an active ingredient an antigen-binding molecule that contains anantigen-binding domain whose antigen-binding activity varies dependingon the concentration of a small molecule compound (e.g., a cancertissue-specific compound, inflammatory tissue-specific compound,metabolites thereof, and such) and has a neutralizing activity against amembrane-type molecule. In another non-limiting embodiment, the presentinvention provides a pharmaceutical composition that induces an immuneresponse, comprising as an active ingredient an antigen-binding moleculethat contains an antigen-binding domain whose antigen-binding activityvaries depending on the concentration of a small molecule compound(e.g., a cancer tissue-specific compound, inflammatory tissue-specificcompound, metabolites thereof, and such) and has a neutralizing activityagainst a membrane-type molecule in addition to a cytotoxic activityagainst cells expressing the membrane-type molecule on their cellmembrane. Generally, a neutralizing activity refers to an activity ofinhibiting the biological activity of a ligand which has a biologicalactivity towards cells, such as viruses and toxins. Thus, a substancehaving a neutralizing activity refers to a substance that binds to aligand or a receptor to which the ligand binds and inhibits the bindingbetween the ligand and the receptor. A receptor whose binding to theligand has been blocked by the neutralizing activity will not be able toexhibit the biological activity through the receptor. When theantigen-binding molecule is an antibody, the antibody having such aneutralizing activity is generally called a neutralizing antibody. Theneutralizing activity of a test substance may be measured by comparingthe biological activities in the presence of a ligand between conditionswhen the test substance is present or absent.

A suitable example of a major ligand for the IL-6 receptor is IL-6,which is shown in SEQ ID NO: 27. The IL-6 receptor, which is an I-typemembrane protein whose amino terminus forms the extracellular domain,forms a hetero-tetramer with the gp130 receptor which was induced byIL-6 to dimerize (Heinrich et al. (Biochem. J. (1998) 334, 297-314)).Formation of the heterotetramer activates Jak associated with the gp130receptor. Jak carries out autophosphorylation and receptorphosphorylation. The phosphorylation sites of the receptor and of Jakserve as binding sites for molecules belonging to the Stat family havingSH2 such as Stat3, and for the MAP kinases, PI3/Akt, and other proteinsand adapters having SH2. Next, Stat that bound to the gp130 receptor isphosphorylated by Jak. The phosphorylated Stat dimerizes andtranslocates to the nucleus, and regulates transcription of targetgenes. Jak and Stat can also be involved in the signaling cascadethrough receptors of other classes. A deregulated IL-6 signaling cascadeis observed in inflammation and pathological conditions of autoimmunediseases, and cancers such as prostate cancer and multiple myeloma.Stat3 which may act as an oncogene is constitutively activated in manycancers. In prostate cancer and multiple myeloma, there is a crosstalkbetween the signaling cascade from the IL-6 receptor and the signalingcascade from members of the epidermal growth factor receptor (EGFR)family (Ishikawa et al. (J. Clin. Exp. Hematopathol. (2006) 46 (2),55-66)).

Such intracellular signaling cascades are different for each cell type;therefore, an appropriate target molecule can be set according to eachof the target cells of interest, and the target molecule is not limitedto the above-mentioned factors. The neutralization activity can beevaluated by measuring the in vivo signal activation. Furthermore,activation of in vivo signals can also be detected by using as anindicator the transcription-inducing action on a target gene that existsdownstream of the in vivo signaling cascade. A change in thetranscription activity of a target gene can be detected by the principleof a reporter assay. Specifically, a reporter gene such as the greenfluorescence protein (GFP) or luciferase is placed downstream of atranscription factor or a promoter region of the target gene; and achange in transcription activity can be measured in terms of reporteractivity by measuring the reporter activity. Commercially available kitsfor measuring in vivo signal activation can be suitably used (forexample, the Mercury Pathway Profiling Luciferase System (Clontech)).

Furthermore, as a method for measuring the neutralization activity on areceptor ligand in the EGF receptor family and such which acts on asignaling cascade that typically works toward enhancing cellproliferation, neutralization activity of an antigen-binding moleculecan be evaluated by measuring the proliferation activity of the targetcells. For example, the following method is suitably used as a methodfor measuring or evaluating inhibitory effects based on theneutralization activity of an anti-HB-EGF antibody against theproliferation of cells whose proliferation is promoted by EGF familygrowth factors such as HB-EGF. As a method for evaluating or measuringthe activity of inhibiting cell proliferation in a test tube, a methodthat measures the incorporation by living cells of [³H]-labeledthymidine added to the culture medium as an index of the DNA replicationability is used. As a more convenient method, a dye exclusion methodthat measures under a microscope the ability of a cell to release a dyesuch as trypan blue to the outside of the cell, or the MTT method isused. The latter makes use of the ability of living cells to convert3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT),which is a tetrazolium salt, to a blue formazan product. Morespecifically, a test antibody is added along with a ligand to theculture solution of a test cell; and after a certain period of time haselapsed, an MTT solution is added to the culture, and this is left tostand for a certain amount of time to let the cell incorporate MTT. As aresult, MTT which is a yellow compound is converted to a blue compoundby succinate dehydrogenase in the mitochondria of the cell. After thisblue product is dissolved for coloration, its absorbance is measured andused as an indicator of the number of viable cells. Besides MTT,reagents such as MTS, XTT, WST-1, and WST-8 are also commerciallyavailable (Nacalai Tesque, and such), and can be suitably used. Formeasurement of the activity, a binding antibody that has the sameisotype as the anti-HB-EGF antibody but does not have the cellproliferation-inhibiting activity can be used as a control antibody inthe same manner as the anti-HB-EGF antibody, and the anti-HB-EGFantibody is judged to have the activity when it shows a stronger cellproliferation-inhibiting activity than the control antibody.

As cells for evaluating activity, for example, cells showingHB-EGF-promoted proliferation such as the RMG-1 cell line which is anovarian cancer cell line may be suitably used; and mouse Ba/F3 cellstransformed with a vector in which a gene encoding hEGFR/mG-CSFR, whichis a fusion protein of the extracellular domain of human EGFR fused inframe with the intracellular domain of the mouse G-CSF receptor, islinked so as to allow expression, may also be suitably used. This way,those skilled in the art may appropriately select cells for evaluatingactivity to measure the cell proliferation activity mentioned above.

Antibody

Herein, “antibody” refers to a natural immunoglobulin or animmunoglobulin produced by partial or complete synthesis. Antibodies canbe isolated from natural sources such as naturally-occurring plasma andserum, or culture supernatants of antibody-producing hybridomas.Alternatively, antibodies can be partially or completely synthesizedusing techniques such as genetic recombination. Preferred antibodiesinclude, for example, antibodies of an immunoglobulin isotype orsubclass belonging thereto. Known human immunoglobulins includeantibodies of the following nine classes (isotypes): IgG1, IgG2, IgG3,IgG4, IgA1, IgA2, IgD, IgE, and IgM. Of these isotypes, antibodies ofthe present invention include IgG1, IgG2, IgG3, and IgG4. A number ofallotype sequences of human IgG1, human IgG2, human IgG3, and human IgG4constant regions due to gene polymorphisms are described in “Sequencesof proteins of immunological interest”, NIH Publication No. 91-3242. Anyof such sequences may be used in the present invention. In particular,for the human IgG1 sequence, the amino acid sequence at positions 356 to358 as indicated by EU numbering may be DEL or EEM. Several allotypesequences due to genetic polymorphisms have been described in “Sequencesof proteins of immunological interest”, NIH Publication No. 91-3242 forthe human Igκ (Kappa) constant region and human Igλ (Lambda) constantregion, and any of the sequences may be used in the present invention.

Methods for producing an antibody with desired binding activity areknown to those skilled in the art. Below is an example that describes amethod for producing an antibody that binds to IL-6R (anti-IL-6Rantibody). Antibodies that bind to an antigen other than IL-6R can alsobe produced according to the example described below.

Anti-IL-6R antibodies can be obtained as polyclonal or monoclonalantibodies using known methods. The anti-IL-6R antibodies preferablyproduced are monoclonal antibodies derived from mammals. Suchmammal-derived monoclonal antibodies include antibodies produced byhybridomas or host cells transformed with an expression vector carryingan antibody gene by genetic engineering techniques. “Humanizedantibodies” or “chimeric antibodies” are included in the monoclonalantibodies of the present invention.

Monoclonal antibody-producing hybridomas can be produced using knowntechniques, for example, as described below. Specifically, mammals areimmunized by conventional immunization methods using an IL-6R protein asa sensitizing antigen. Resulting immune cells are fused with knownparental cells by conventional cell fusion methods. Then, hybridomasproducing an anti-IL-6R antibody can be selected by screening formonoclonal antibody-producing cells using conventional screeningmethods.

Specifically, monoclonal antibodies are prepared as mentioned below.First, the IL-6R gene whose nucleotide sequence is disclosed in SEQ IDNO: 2 can be expressed to produce an IL-6R protein shown in SEQ ID NO:1, which will be used as a sensitizing antigen for antibody preparation.That is, a gene sequence encoding IL-6R is inserted into a knownexpression vector, and appropriate host cells are transformed with thisvector. The desired human IL-6R protein is purified from the host cellsor their culture supernatants by known methods. In order to obtainsoluble IL-6R from culture supernatants, for example, a proteinconsisting of the amino acids at positions 1 to 357 in the IL-6Rpolypeptide sequence of SEQ ID NO: 1, such as described in Mullberg etal. (J. Immunol. (1994) 152 (10), 4958-4968), is expressed as a solubleIL-6R, instead of the IL-6R protein of SEQ ID NO: 1. Purified naturalIL-6R protein can also be used as a sensitizing antigen.

The purified IL-6R protein can be used as a sensitizing antigen forimmunization of mammals. A partial IL-6R peptide may also be used as asensitizing antigen. In this case, a partial peptide can be prepared bychemical synthesis based on the amino acid sequence of human IL-6R, orby inserting a partial IL-6R gene into an expression vector forexpression. Alternatively, a partial peptide can be produced bydegrading an IL-6R protein with a protease. The length and region of thepartial IL-6R peptide are not limited to particular embodiments. Apreferred region can be arbitrarily selected from the amino acidsequence at amino acid positions 20 to 357 in the amino acid sequence ofSEQ ID NO: 1. The number of amino acids forming a peptide to be used asa sensitizing antigen is preferably at least five or more, six or more,or seven or more. More specifically, a peptide of 8 to 50 residues, morepreferably 10 to 30 residues can be used as a sensitizing antigen.

For sensitizing antigen, alternatively it is possible to use a fusionprotein prepared by fusing a desired partial polypeptide or peptide ofthe IL-6R protein with a different polypeptide. For example, antibody Fcfragments and peptide tags are preferably used to produce fusionproteins to be used as sensitizing antigens. Vectors for expression ofsuch fusion proteins can be constructed by fusing in frame genesencoding two or more desired polypeptide fragments and inserting thefusion gene into an expression vector as described above. Methods forproducing fusion proteins are described in Molecular Cloning 2nd ed.(Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58 (1989) ColdSpring Harbor Lab. Press). Methods for preparing IL-6R to be used as asensitizing antigen, and immunization methods using IL-6R arespecifically described in WO 2003/000883, WO 2004/022754, WO2006/006693, and such.

There is no particular limitation on the mammals to be immunized withthe sensitizing antigen. However, it is preferable to select the mammalsby considering their compatibility with the parent cells to be used forcell fusion. In general, rodents such as mice, rats, and hamsters,rabbits, and monkeys are preferably used.

The above animals are immunized with a sensitizing antigen by knownmethods. Generally performed immunization methods include, for example,intraperitoneal or subcutaneous injection administration of asensitizing antigen into mammals. Specifically, a sensitizing antigen isappropriately diluted with PBS (Phosphate-Buffered Saline),physiological saline, or the like. If desired, a conventional adjuvantsuch as Freund's complete adjuvant is mixed with the antigen, and themixture is emulsified. Then, the sensitizing antigen is administered toa mammal several times at 4- to 21-day intervals. Appropriate carriersmay be used in immunization with the sensitizing antigen. In particular,when a low-molecular-weight partial peptide is used as the sensitizingantigen, it is sometimes desirable to couple the sensitizing antigenpeptide to a carrier protein such as albumin or keyhole limpethemocyanin for immunization.

Alternatively, hybridomas producing a desired antibody can be preparedusing DNA immunization as mentioned below. DNA immunization is animmunization method that confers immunostimulation by expressing asensitizing antigen in an animal immunized as a result of administeringa vector DNA constructed to allow expression of an antigenprotein-encoding gene in the animal. As compared to conventionalimmunization methods in which a protein antigen is administered toanimals to be immunized, DNA immunization is expected to be superior inthat:

immunostimulation can be provided while retaining the structure of amembrane protein such as IL-6R; and

there is no need to purify the antigen for immunization.

In order to prepare a monoclonal antibody of the present invention usingDNA immunization, first, a DNA expressing an IL-6R protein isadministered to an animal to be immunized. The IL-6R-encoding DNA can besynthesized by known methods such as PCR. The obtained DNA is insertedinto an appropriate expression vector, and then this is administered toan animal to be immunized. Preferably used expression vectors include,for example, commercially-available expression vectors such as pcDNA3.1.Vectors can be administered to an organism using conventional methods.For example, DNA immunization is performed by using a gene gun tointroduce expression vector-coated gold particles into cells in the bodyof an animal to be immunized. Antibodies that recognized IL-6R can alsobe produced by the methods described in WO 2003/104453.

After immunizing a mammal as described above, an increase in the titerof an IL-6R-binding antibody is confirmed in the serum. Then, immunecells are collected from the mammal, and then subjected to cell fusion.In particular, splenocytes are preferably used as immune cells.

A mammalian myeloma cell is used as a cell to be fused with theabove-mentioned immune cells. The myeloma cells preferably comprise asuitable selection marker for screening. A selection marker conferscharacteristics to cells for their survival (or death) under a specificculture condition. Hypoxanthine-guanine phosphoribosyltransferasedeficiency (hereinafter abbreviated as HGPRT deficiency) and thymidinekinase deficiency (hereinafter abbreviated as TK deficiency) are knownas selection markers. Cells with HGPRT or TK deficiency havehypoxanthine-aminopterin-thymidine sensitivity (hereinafter abbreviatedas HAT sensitivity). HAT-sensitive cells cannot synthesize DNA in a HATselection medium, and are thus killed. However, when the cells are fusedwith normal cells, they can continue DNA synthesis using the salvagepathway of the normal cells, and therefore they can grow even in the HATselection medium.

HGPRT-deficient and TK-deficient cells can be selected in a mediumcontaining 6-thioguanine, 8-azaguanine (hereinafter abbreviated as 8AG),or 5′-bromodeoxyuridine, respectively. Normal cells are killed becausethey incorporate these pyrimidine analogs into their DNA. Meanwhile,cells that are deficient in these enzymes can survive in the selectionmedium, since they cannot incorporate these pyrimidine analogs. Inaddition, a selection marker referred to as G418 resistance provided bythe neomycin-resistant gene confers resistance to 2-deoxystreptamineantibiotics (gentamycin analogs). Various types of myeloma cells thatare suitable for cell fusion are known.

For example, myeloma cells including the following cells can bepreferably used:

P3(P3x63Ag8.653) (J. Immunol. (1979) 123 (4), 1548-1550); P3x63Ag8U.1(Current Topics in Microbiology and Immunology (1978) 81, 1-7);

NS-1 (C. Eur. J. Immunol. (1976) 6 (7), 511-519);

MPC-11 (Cell (1976) 8 (3), 405-415); SP2/0 (Nature (1978) 276 (5685),269-270);

FO (J. Immunol. Methods (1980) 35 (1-2), 1-21);S194/5.XXO.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323);

R210 (Nature (1979) 277 (5692), 131-133), etc.

Cell fusions between the immunocytes and myeloma cells are essentiallycarried out using known methods, for example, a method by Kohler andMilstein et al. (Methods Enzymol. (1981) 73: 3-46).

More specifically, cell fusion can be carried out, for example, in aconventional culture medium in the presence of a cell fusion-promotingagent. The fusion-promoting agents include, for example, polyethyleneglycol (PEG) and Sendai virus (HVJ). If required, an auxiliary substancesuch as dimethyl sulfoxide is also added to improve fusion efficiency.

The ratio of immune cells to myeloma cells may be determined at one'sown discretion, preferably, for example, one myeloma cell for every oneto ten immunocytes. Culture media to be used for cell fusions include,for example, media that are suitable for the growth of myeloma celllines, such as RPMI1640 medium and MEM medium, and other conventionalculture medium used for this type of cell culture. In addition, serumsupplements such as fetal calf serum (FCS) may be preferably added tothe culture medium.

For cell fusion, predetermined amounts of the above immune cells andmyeloma cells are mixed well in the above culture medium. Then, a PEGsolution (for example, the average molecular weight is about 1,000 to6,000) prewarmed to about 37° C. is added thereto at a concentration ofgenerally 30% to 60% (w/v). This is gently mixed to produce desiredfusion cells (hybridomas). Then, an appropriate culture medium mentionedabove is gradually added to the cells, and this is repeatedlycentrifuged to remove the supernatant. Thus, cell fusion agents and suchwhich are unfavorable to hybridoma growth can be removed.

The hybridomas thus obtained can be selected by culture using aconventional selective medium, for example, HAT medium (a culture mediumcontaining hypoxanthine, aminopterin, and thymidine). Cells other thanthe desired hybridomas (non-fused cells) can be killed by continuingculture in the above HAT medium for a sufficient period of time.Typically, the period is several days to several weeks. Then, hybridomasproducing the desired antibody are screened and singly cloned byconventional limiting dilution methods.

The hybridomas thus obtained can be selected using a selection mediumbased on the selection marker possessed by the myeloma used for cellfusion. For example, HGPRT- or TK-deficient cells can be selected byculture using the HAT medium (a culture medium containing hypoxanthine,aminopterin, and thymidine). Specifically, when HAT-sensitive myelomacells are used for cell fusion, cells successfully fused with normalcells can selectively proliferate in the HAT medium. Cells other thanthe desired hybridomas (non-fused cells) can be killed by continuingculture in the above HAT medium for a sufficient period of time.Specifically, desired hybridomas can be selected by culture forgenerally several days to several weeks. Then, hybridomas producing thedesired antibody are screened and singly cloned by conventional limitingdilution methods.

Desired antibodies can be preferably selected and singly cloned byscreening methods based on known antigen/antibody reaction. For example,an IL-6R-binding monoclonal antibody can bind to IL-6R expressed on thecell surface. Such a monoclonal antibody can be screened by fluorescenceactivated cell sorting (FACS). FACS is a system that assesses thebinding of an antibody to cell surface by analyzing cells contacted witha fluorescent antibody using laser beam, and measuring the fluorescenceemitted from individual cells.

To screen for hybridomas that produce a monoclonal antibody of thepresent invention by FACS, IL-6R-expressing cells are first prepared.Cells preferably used for screening are mammalian cells in which IL-6Ris forcedly expressed. As control, the activity of an antibody to bindto cell-surface IL-6R can be selectively detected using non-transformedmammalian cells as host cells. Specifically, hybridomas producing ananti-IL-6R monoclonal antibody can be isolated by selecting hybridomasthat produce an antibody which binds to cells forced to express IL-6R,but not to host cells.

Alternatively, the activity of an antibody to bind to immobilizedIL-6R-expressing cells can be assessed based on the principle of ELISA.For example, IL-6R-expressing cells are immobilized to the wells of anELISA plate. Culture supernatants of hybridomas are contacted with theimmobilized cells in the wells, and antibodies that bind to theimmobilized cells are detected. When the monoclonal antibodies arederived from mouse, antibodies bound to the cells can be detected usingan anti-mouse immunoglobulin antibody. Hybridomas producing a desiredantibody having the antigen-binding ability are selected by the abovescreening, and they can be cloned by a limiting dilution method or thelike.

Monoclonal antibody-producing hybridomas thus prepared can be passagedin a conventional culture medium, and stored in liquid nitrogen for along period.

The above hybridomas are cultured by a conventional method, and desiredmonoclonal antibodies can be prepared from the culture supernatants.Alternatively, the hybridomas are administered to and grown incompatible mammals, and monoclonal antibodies are prepared from theascites. The former method is suitable for preparing antibodies withhigh purity.

Antibodies encoded by antibody genes that are cloned fromantibody-producing cells such as the above hybridomas can also bepreferably used. A cloned antibody gene is inserted into an appropriatevector, and this is introduced into a host to express the antibodyencoded by the gene. Methods for isolating antibody genes, inserting thegenes into vectors, and transforming host cells have already beenestablished, for example, by Vandamme et al. (Eur. J. Biochem. (1990)192(3), 767-775). Methods for producing recombinant antibodies are alsoknown as described below.

For example, a cDNA encoding the variable region (V region) of ananti-IL-6R antibody is prepared from hybridoma cells expressing theanti-IL-6R antibody. For this purpose, total RNA is first extracted fromhybridomas. Methods used for extracting mRNAs from cells include, forexample:

the guanidine ultracentrifugation method (Biochemistry (1979) 18(24),5294-5299), and

the AGPC method (Anal. Biochem. (1987) 162(1), 156-159)

Extracted mRNAs can be purified using the mRNA Purification Kit (GEHealthcare Bioscience) or such. Alternatively, kits for extracting totalmRNA directly from cells, such as the QuickPrep mRNA Purification Kit(GE Healthcare Bioscience), are also commercially available. mRNAs canbe prepared from hybridomas using such kits. cDNAs encoding the antibodyV region can be synthesized from the prepared mRNAs using a reversetranscriptase. cDNAs can be synthesized using the AMV ReverseTranscriptase First-strand cDNA Synthesis Kit (Seikagaku Co.) or such.Furthermore, the SMART RACE cDNA amplification kit (Clontech) and thePCR-based 5′-RACE method (Proc. Natl. Acad. Sci. U.S.A. (1988) 85(23),8998-9002; Nucleic Acids Res. (1989) 17(8), 2919-2932) can beappropriately used to synthesize and amplify cDNAs. In such a cDNAsynthesis process, appropriate restriction enzyme sites described belowmay be introduced into both ends of a cDNA.

The cDNA fragment of interest is purified from the resulting PCRproduct, and then this is ligated to a vector DNA. A recombinant vectoris thus constructed, and introduced into E. coli or such. After colonyselection, the desired recombinant vector can be prepared from thecolony-forming E. coli. Then, whether the recombinant vector has thecDNA nucleotide sequence of interest is tested by a known method such asthe dideoxy nucleotide chain termination method.

The 5′-RACE method which uses primers to amplify the variable regiongene is conveniently used for isolating the gene encoding the variableregion. First, a 5′-RACE cDNA library is constructed by cDNA synthesisusing RNAs extracted from hybridoma cells as a template. A commerciallyavailable kit such as the SMART RACE cDNA amplification kit isappropriately used to synthesize the 5′-RACE cDNA library.

The antibody gene is amplified by PCR using the prepared 5′-RACE cDNAlibrary as a template. Primers for amplifying the mouse antibody genecan be designed based on known antibody gene sequences. The nucleotidesequences of the primers vary depending on the immunoglobulin subclass.Therefore, it is preferable that the subclass is determined in advanceusing a commercially available kit such as the Iso Strip mousemonoclonal antibody isotyping kit (Roche Diagnostics).

Specifically, for example, primers that allow amplification of genesencoding γ1, γ2a, γ2b, and γ3 heavy chains and κ and λ light chains areused to isolate mouse IgG-encoding genes. In general, a primer thatanneals to a constant region site close to the variable region is usedas a 3′-side primer to amplify an IgG variable region gene. Meanwhile, aprimer attached to a 5′ RACE cDNA library construction kit is used as a5′-side primer.

PCR products thus amplified are used to reshape immunoglobulins composedof a combination of heavy and light chains. A desired antibody can beselected using the IL-6R-binding activity of a reshaped immunoglobulinas an indicator. For example, when the objective is to isolate anantibody against IL-6R, it is more preferred that the binding of theantibody to IL-6R is specific. An IL-6R-binding antibody can bescreened, for example, by the following steps:

(1) contacting an IL-6R-expressing cell with an antibody comprising theV region encoded by a cDNA isolated from a hybridoma;

(2) detecting the binding of the antibody to the IL-6R-expressing cell;and

(3) selecting an antibody that binds to the IL-6R-expressing cell.

Methods for detecting the binding of an antibody to IL-6R-expressingcells are known. Specifically, the binding of an antibody toIL-6R-expressing cells can be detected by the above-described techniquessuch as FACS. Immobilized samples of IL-6R-expressing cells areappropriately used to assess the binding activity of an antibody.

Preferred antibody screening methods that use the binding activity as anindicator also include panning methods using phage vectors. Screeningmethods using phage vectors are advantageous when the antibody genes areisolated from heavy-chain and light-chain subclass libraries from apolyclonal antibody-expressing cell population. Genes encoding theheavy-chain and light-chain variable regions can be linked by anappropriate linker sequence to form a single-chain Fv (scFv). Phagespresenting scFv on their surface can be produced by inserting a geneencoding scFv into a phage vector. The phages are contacted with anantigen of interest. Then, a DNA encoding scFv having the bindingactivity of interest can be isolated by collecting phages bound to theantigen. This process can be repeated as necessary to enrich scFv havingthe binding activity of interest.

After isolation of the cDNA encoding the V region of the anti-IL-6Rantibody of interest, the cDNA is digested with restriction enzymes thatrecognize the restriction sites introduced into both ends of the cDNA.Preferred restriction enzymes recognize and cleave a nucleotide sequencethat occurs in the nucleotide sequence of the antibody gene at a lowfrequency. Furthermore, a restriction site for an enzyme that produces asticky end is preferably introduced into a vector to insert asingle-copy digested fragment in the correct orientation. The cDNAencoding the V region of the anti-IL-6R antibody is digested asdescribed above, and this is inserted into an appropriate expressionvector to construct an antibody expression vector. In this case, if agene encoding the antibody constant region (C region) and a geneencoding the above V region are fused in-frame, a chimeric antibody isobtained. Herein, “chimeric antibody” means that the origin of theconstant region is different from that of the variable region. Thus, inaddition to mouse/human heterochimeric antibodies, human/humanallochimeric antibodies are included in the chimeric antibodies of thepresent invention. A chimeric antibody expression vector can beconstructed by inserting the above V region gene into an expressionvector that already has the constant region. Specifically, for example,a recognition sequence for a restriction enzyme that excises the above Vregion gene can be appropriately placed on the 5′ side of an expressionvector carrying a DNA encoding a desired antibody constant region. Achimeric antibody expression vector is constructed by fusing in framethe two genes digested with the same combination of restriction enzymes.

To produce an anti-IL-6R monoclonal antibody, antibody genes areinserted into an expression vector so that the genes are expressed underthe control of an expression regulatory region. The expressionregulatory region for antibody expression includes, for example,enhancers and promoters. Furthermore, an appropriate signal sequence maybe attached to the amino terminus so that the expressed antibody issecreted to the outside of cells. In the Examples below, a peptidehaving the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 3) isused as a signal sequence. Meanwhile, other appropriate signal sequencesmay be attached. The expressed polypeptide is cleaved at the carboxylterminus of the above sequence, and the resulting polypeptide issecreted to the outside of cells as a mature polypeptide. Then,appropriate host cells are transformed with the expression vector, andrecombinant cells expressing the anti-IL-6R antibody-encoding DNA areobtained.

DNAs encoding the antibody heavy chain (H chain) and light chain (Lchain) are separately inserted into different expression vectors toexpress the antibody gene. An antibody molecule having the H and Lchains can be expressed by co-transfecting the same host cell withvectors into which the H-chain and L-chain genes are respectivelyinserted. Alternatively, host cells can be transformed with a singleexpression vector into which DNAs encoding the H and L chains areinserted (see WO 1994/011523).

There are various known host cell/expression vector combinations forantibody preparation by introducing isolated antibody genes intoappropriate hosts. All of these expression systems are applicable toisolation of the antigen-binding domains of the present invention.Appropriate eukaryotic cells used as host cells include animal cells,plant cells, and fungal cells. Specifically, the animal cells include,for example, the following cells.

(1) mammalian cells: CHO (Chinese hamster ovary cell line), COS (Monkeykidney cell line), myeloma (Sp2/0, NS0, etc.), BHK (baby hamster kidneycell line), HeLa, Vero, HEK293 (human embryonic kidney cell line withsheared adenovirus (Ad)5 DNA), PER. C6 cell (human embryonic retinalcell line transformed with the Adenovirus Type 5 (Ad5) E1A and E1Bgenes) and such (Current Protocols in Protein Science (May, 2001, Unit5.9, Table 5.9.1));(2) amphibian cells: Xenopus oocytes, or such; and(3) insect cells: sf9, sf21, Tn5, or such.

In addition, as a plant cell, an antibody gene expression system usingcells derived from the Nicotiana genus such as Nicotiana tabacum isknown. Callus cultured cells can be appropriately used to transformplant cells.

Furthermore, the following cells can be used as fungal cells:

yeasts: the Saccharomyces genus such as Saccharomyces cerevisiae, andthe Pichia genus such as Pichia pastoris; andfilamentous fungi: the Aspergillus genus such as Aspergillus niger.

Furthermore, antibody gene expression systems that utilize prokaryoticcells are also known. For example, when using bacterial cells, E. colicells, Bacillus subtilis cells, and such can suitably be utilized in thepresent invention. Expression vectors carrying the antibody genes ofinterest are introduced into these cells by transfection. Thetransfected cells are cultured in vitro, and the desired antibody can beprepared from the culture of transformed cells.

In addition to the above-described host cells, transgenic animals canalso be used to produce a recombinant antibody. That is, the antibodycan be obtained from an animal into which the gene encoding the antibodyof interest is introduced. For example, the antibody gene can beconstructed as a fusion gene by inserting in frame into a gene thatencodes a protein produced specifically in milk. Goat β-casein or suchcan be used, for example, as the protein secreted in milk. DNA fragmentscontaining the fused gene inserted with the antibody gene is injectedinto a goat embryo, and then this embryo is introduced into a femalegoat. Desired antibodies can be obtained as a protein fused with themilk protein from milk produced by the transgenic goat born from theembryo-recipient goat (or progeny thereof). In addition, to increase thevolume of milk containing the desired antibody produced by thetransgenic goat, hormones can be administered to the transgenic goat asnecessary (Bio/Technology (1994) 12 (7), 699-702).

When an antigen-binding molecule described herein is administered tohuman, an antigen-binding domain derived from a genetically recombinantantibody that has been artificially altered to reduce the heterologousantigenicity against human and such, can be appropriately used as theantigen-binding domain of the antigen-binding molecule. Such geneticallyrecombinant antibodies include, for example, humanized antibodies. Thesealtered antibodies are appropriately produced by known methods.

An antibody variable region used to produce the antigen-binding domainof an antigen-binding molecule described herein is generally formed bythree complementarity-determining regions (CDRs) that are separated byfour framework regions (FRs). CDR is a region that substantiallydetermines the binding specificity of an antibody. The amino acidsequences of CDRs are highly diverse. On the other hand, the FR-formingamino acid sequences often have high identity even among antibodies withdifferent binding specificities. Therefore, generally, the bindingspecificity of a certain antibody can be introduced to another antibodyby CDR grafting.

A humanized antibody is also called a reshaped human antibody.Specifically, humanized antibodies prepared by grafting the CDR of anon-human animal antibody such as a mouse antibody to a human antibodyand such are known. Common genetic engineering techniques for obtaininghumanized antibodies are also known. Specifically, for example, overlapextension PCR is known as a method for grafting a mouse antibody CDR toa human FR. In overlap extension PCR, a nucleotide sequence encoding amouse antibody CDR to be grafted is added to primers for synthesizing ahuman antibody FR. Primers are prepared for each of the four FRs. It isgenerally considered that when grafting a mouse CDR to a human FR,selecting a human FR that has high identity to a mouse FR isadvantageous for maintaining the CDR function. That is, it is generallypreferable to use a human FR comprising an amino acid sequence which hashigh identity to the amino acid sequence of the FR adjacent to the mouseCDR to be grafted.

Nucleotide sequences to be ligated are designed so that they will beconnected to each other in frame. Human FRs are individually synthesizedusing the respective primers. As a result, products in which the mouseCDR-encoding DNA is attached to the individual FR-encoding DNAs areobtained. Nucleotide sequences encoding the mouse CDR of each productare designed so that they overlap with each other. Then, complementarystrand synthesis reaction is conducted to anneal the overlapping CDRregions of the products synthesized using a human antibody gene astemplate. Human FRs are ligated via the mouse CDR sequences by thisreaction.

The full length V region gene, in which three CDRs and four FRs areultimately ligated, is amplified using primers that anneal to its 5′- or3′-end, which are added with suitable restriction enzyme recognitionsequences. An expression vector for humanized antibody can be producedby inserting the DNA obtained as described above and a DNA that encodesa human antibody C region into an expression vector so that they willligate in frame. After the recombinant vector is transfected into a hostto establish recombinant cells, the recombinant cells are cultured, andthe DNA encoding the humanized antibody is expressed to produce thehumanized antibody in the cell culture (see, European Patent PublicationNo. EP 239400 and International Patent Publication No. WO 1996/002576).

By qualitatively or quantitatively measuring and evaluating theantigen-binding activity of the humanized antibody produced as describedabove, one can suitably select human antibody FRs that allow CDRs toform a favorable antigen-binding site when ligated through the CDRs.Amino acid residues in FRs may be substituted as necessary, so that theCDRs of a reshaped human antibody form an appropriate antigen-bindingsite. For example, amino acid sequence mutations can be introduced intoFRs by applying the PCR method used for grafting a mouse CDR into ahuman FR. More specifically, partial nucleotide sequence mutations canbe introduced into primers that anneal to the FR. Nucleotide sequencemutations are introduced into the FRs synthesized by using such primers.Mutant FR sequences having the desired characteristics can be selectedby measuring and evaluating the activity of the amino acid-substitutedmutant antibody to bind to the antigen by the above-mentioned method(Cancer Res. (1993) 53: 851-856).

Alternatively, desired human antibodies can be obtained by immunizingtransgenic animals having the entire repertoire of human antibody genes(see WO 1993/012227; WO 1992/003918; WO 1994/002602; WO 1994/025585; WO1996/034096; WO 1996/033735) by DNA immunization.

Furthermore, techniques for preparing human antibodies by panning usinghuman antibody libraries are also known. For example, the V region of ahuman antibody is expressed as a single-chain antibody (scFv) on phagesurface by the phage display method. Phages expressing an scFv thatbinds to the antigen can be selected. The DNA sequence encoding thehuman antibody V region that binds to the antigen can be determined byanalyzing the genes of selected phages. The DNA sequence of the scFvthat binds to the antigen is determined. An expression vector isprepared by fusing the V region sequence in frame with the C regionsequence of a desired human antibody, and inserting this into anappropriate expression vector. The expression vector is introduced intocells appropriate for expression such as those described above. Thehuman antibody can be produced by expressing the human antibody-encodinggene in the cells. These methods are already known (see WO 1992/001047;WO 1992/020791; WO 1993/006213; WO 1993/011236; WO 1993/019172; WO1995/001438; WO 1995/015388).

Besides the phage display method, techniques of using a cell-freetranslation system, techniques of displaying antigen-binding moleculeson the surface of cells or viruses, techniques of using emulsions, andsuch are known as techniques for obtaining human antibodies by panningusing a human antibody library. As techniques of using a cell-freetranslation system, for example, the ribosome display method where acomplex is formed between an mRNA and the translated protein via theribosome by removing the stop codon and such, the cDNA display methodwhere a gene sequence and the translated protein are covalently linkedusing a compound such as puromycin, the mRNA display method, the CISdisplay method where a complex is formed between a gene and thetranslated protein using a nucleic acid-binding protein, or such may beused. For techniques of presenting an antigen-binding molecule on thesurface of cells or viruses, the E. coli display method, Gram-positivebacterium display method, yeast display method, mammalian cell displaymethod, virus display method, and such may be used besides the phagedisplay method. As techniques that use emulsions, the in vitro virusdisplay method which involves incorporating genes andtranslation-related molecules into an emulsion, and such may be used.These methods are already publicly known (Nat Biotechnol. 2000 December;18(12): 1287-92, Nucleic Acids Res. 2006; 34(19):e127, Proc Natl AcadSci USA. 2004 Mar. 2; 101(9):2806-10, Proc Natl Acad Sci USA. 2004 Jun.22; 101(25):9193-8, Protein Eng Des Sel. 2008 April; 21(4):247-55, ProcNatl Acad Sci USA. 2000 Sep. 26; 97(20):10701-5, MAbs. 2010September-October; 2(5):508-18, Methods Mol Biol. 2012; 911:183-98).

In addition to the techniques described above, techniques of B cellcloning (identification of each antibody-encoding sequence, cloning andits isolation; use in constructing expression vector in order to prepareeach antibody (IgG1, IgG2, IgG3, or IgG4 in particular); and such) suchas described in Bernasconi et al. (Science (2002) 298: 2199-2202) or inWO 2008/081008 can be appropriately used to isolate antibody genes.

A non-limiting embodiment of antibodies in the present inventionincludes but is not limited to chimeric antigen receptors that areincorporated into T-cells, which are fusions of an antibody or fragmentsthereof that recognize antigens instead of a T-cell receptor and T-cellsignal domains, as well as T-cells into which the chimeric antigenreceptor has been incorporated.

EU Numbering and Kabat Numbering

According to the methods used in the present invention, amino acidpositions assigned to antibody CDR and FR are specified according toKabat's numbering (Sequences of Proteins of Immunological Interest(National Institute of Health, Bethesda, Md., 1987 and 1991)). Herein,when an antigen-binding molecule is an antibody or antigen-bindingfragment, variable region amino acids are indicated by Kabat numbering,while constant region amino acids are indicated by EU numbering based onKabat's amino acid positions.

Antigen-Binding Domain Whose Binding Activity Varies Depending on theConcentration of a Small Molecule Compound

Examples of a small molecule compound include target tissue-specificcompounds and unnatural compounds. Examples of a method for selectingantigen-binding domains dependently on a target tissue-specific compoundare shown below; and methods such as those for selecting antigen-bindingdomains dependently on a small molecule compound other than targettissue-specific compounds, and the like, may also be carried outappropriately according to the examples below. To obtain anantigen-binding domain (or an antigen-binding molecule containing thedomain) whose antigen-binding activity varies depending on theconcentration of a target tissue-specific compound, the methodsindicated in the above section on binding activity may be appropriatelyapplied. As a non-limiting embodiment, some specific examples of themethods are presented below. For example, to confirm that theantigen-binding activity of an antigen-binding domain (or anantigen-binding molecule containing the domain) in the presence of atarget tissue-specific compound becomes higher than the antigen-bindingactivity of an antigen-binding domain (or an antigen-binding moleculecontaining the domain) in the absence of the compound, theantigen-binding activities of the antigen-binding domain (or theantigen-binding molecule containing the domain) in the presence andabsence of the target tissue-specific compound or in the presence ofhigh and low concentrations of the compound are compared. In anothernon-limiting embodiment, for example, to confirm that theantigen-binding activity of an antigen-binding domain (or anantigen-binding molecule containing the domain) in the presence of ahigh concentration of a target tissue-specific compound becomes higherthan the antigen-binding activity of an antigen-binding domain (or anantigen-binding molecule containing the domain) in the presence of a lowconcentration of the compound, the antigen-binding activities of theantigen-binding domain (or the antigen-binding molecule containing thedomain) in the presence of high and low concentrations of the targettissue-specific compound are compared.

Furthermore, in the present invention, the phrase “the antigen-bindingactivity in the presence of a target tissue-specific compound is higherthan the antigen-binding activity in the absence of the compound” can bealternatively expressed as “the antigen-binding activity of anantigen-binding domain (or an antigen-binding molecule containing thedomain) in the absence of a target tissue-specific compound is lowerthan the antigen-binding activity in the presence of the compound”.Furthermore, in the present invention, “the antigen-binding activity ofan antigen-binding domain (or an antigen-binding molecule containing thedomain) in the absence of a target tissue-specific compound is lowerthan the antigen-binding activity in the presence of the compound” maybe alternatively described as “the antigen-binding activity of anantigen-binding domain (or an antigen-binding molecule containing thedomain) in the absence of a target tissue-specific compound is weakerthan the antigen-binding activity in the presence of the compound”.

Furthermore, in the present invention, the phrase “the antigen-bindingactivity in the presence of a high concentration of a targettissue-specific compound is higher than the antigen-binding activity inthe presence of a low concentration of the compound” can bealternatively expressed as “the antigen-binding activity of anantigen-binding domain (or an antigen-binding molecule containing thedomain) in the presence of a low concentration of a targettissue-specific compound is lower than the antigen-binding activity inthe presence of a high concentration of the compound”. In the presentinvention, “the antigen-binding activity of an antigen-binding domain(or an antigen-binding molecule containing the domain) in the presenceof a low concentration of a target tissue-specific compound is lowerthan the antigen-binding activity in the presence of a highconcentration of the compound” may be alternatively described as “theantigen-binding activity of an antigen-binding domain (or anantigen-binding molecule containing the domain) in the presence of a lowconcentration of a target tissue-specific compound is weaker than theantigen-binding activity in the presence of a high concentration of thecompound”.

Conditions when measuring antigen-binding activity other than theconcentration of a target tissue-specific compound are not particularlylimited, and can be selected appropriately by those skilled in the art.For example, it is possible to measure under conditions of HEPES bufferand 37° C. For example, Biacore (GE Healthcare) or such can be used formeasurement. When the antigen is a soluble molecule, the activity of anantigen-binding domain (or an antigen-binding molecule containing thedomain) to bind to the soluble molecule can be determined by loading theantigen as an analyte onto a chip immobilized with the antigen-bindingdomain (or an antigen-binding molecule containing the domain).Alternatively, when the antigen is a membrane-type molecule, the bindingactivity towards the membrane-type molecule can be determined by loadingthe antigen-binding domain (or an antigen-binding molecule containingthe domain) as an analyte onto a chip immobilized with the antigen.

As long as the antigen-binding activity of an antigen-binding domain (oran antigen-binding molecule containing the domain) contained inantigen-binding molecules of the present invention in the absence of atarget tissue-specific compound is weaker than the antigen-bindingactivity in the presence of the target tissue-specific compound, theratio between the antigen-binding activity in the absence of thecompound and the antigen-binding activity in the presence of thecompound is not particularly limited. However, the value of KD (in theabsence of the compound)/KD (in the presence of the compound), which isa ratio of dissociation constant (KD) against an antigen in the absenceof the target tissue-specific compound to KD in the presence of thecompound, is preferably 2 or greater, more preferably 10 or greater, andstill more preferably 40 or greater. The upper limit of the value of KD(in the absence of the compound)/KD (in the presence of the compound) isnot particularly limited, and may be any value, for example, 400, 1,000,or 10,000, as long as it can be provided by the technologies of thoseskilled in the art. When antigen-binding activity is not observed in theabsence of the target tissue-specific compound, the value of the upperlimit is infinity.

As long as the antigen-binding activity of an antigen-binding domain (oran antigen-binding molecule containing the domain) contained inantigen-binding molecules of the present invention in the presence of alow concentration of a target tissue-specific is weaker than theantigen-binding activity in the presence of a high concentration of thetarget tissue-specific compound, the ratio between the antigen-bindingactivity in the presence of a low concentration of the compound and theantigen-binding activity in the presence of a high concentration of thecompound is not particularly limited. However, the value of KD (in thepresence of a low concentration of the compound)/KD (in the presence ofa high concentration of the compound), which is a ratio of dissociationconstant (KD) against an antigen in the presence of a low concentrationof the target tissue-specific compound to KD in the presence of a highconcentration of the compound, is preferably 2 or greater, morepreferably 10 or greater, and still more preferably 40 or greater. Theupper limit of the value of KD (in the presence of a low concentrationof the compound)/KD (in the presence of a high concentration of thecompound) is not particularly limited, and may be any value, forexample, 400, 1,000, or 10,000, as long as it can be provided by thetechnologies of those skilled in the art. When antigen-binding activityis not observed in the presence of a low concentration of the targettissue-specific compound, the value of the upper limit is infinity.

For the value of antigen-binding activity, if the antigen is a solublemolecule, dissociation constant (KD) can be used; and if the antigen isa membrane-type molecule, apparent dissociation constant (apparent KD)can be used. The dissociation constant (KD) and apparent dissociationconstant (apparent KD) can be determined by methods known to thoseskilled in the art, for example, using Biacore (GE Healthcare), aScatchard plot, a flow cytometer, or such.

As another indicator that shows the ratio between the antigen-bindingactivity of an antigen-binding domain (or an antigen-binding moleculecontaining the domain) of the present invention in the absence of atarget tissue-specific compound and the antigen-binding activity in thepresence of the compound, for example, dissociation rate constant kd canbe suitably used. When the dissociation rate constant (kd) is usedinstead of the dissociation constant (KD) as an indicator that shows thebinding activity ratio, the value of kd (in the absence of thecompound)/kd (in the presence of the compound), which is a ratio betweenkd (dissociation rate constant) for an antigen in the absence of atarget tissue-specific compound and kd in the presence of the compound,is preferably 2 or greater, more preferably 5 or greater, even morepreferably 10 or greater, and still more preferably 30 or greater. Theupper limit of the value of kd (in the absence of the compound)/kd (inthe presence of the compound) is not particularly limited, and may beany value, for example, 50, 100, or 200, as long as it can be providedby the common technical knowledge of those skilled in the art. Whenantigen-binding activity is not observed in the absence of thetissue-specific compound, there is no dissociation and the value of theupper limit becomes infinity.

As another indicator that shows the ratio between the antigen-bindingactivity of an antigen-binding domain (or an antigen-binding moleculecontaining the domain) of the present invention in the presence of a lowconcentration of a target tissue-specific compound and theantigen-binding activity in the presence of a high concentration of thecompound, for example, dissociation rate constant kd can be suitablyused. When the dissociation rate constant (kd) is used instead of thedissociation constant (KD) as an indicator showing the binding activityratio, the value of kd (in the presence of a low concentration of thecompound)/kd (in the presence of a high concentration of the compound),which is a ratio between kd (dissociation rate constant) for an antigenin the presence of a low concentration of a target tissue-specificcompound and kd in the presence of a high concentration of the compound,is preferably 2 or greater, more preferably 5 or greater, even morepreferably 10 or greater, and still more preferably 30 or greater. Theupper limit of the value of kd (in the presence of a low concentrationof the compound)/kd (in the presence of a high concentration of thecompound) is not particularly limited, and may be any value, forexample, 50, 100, or 200, as long as it can be provided by the commontechnical knowledge of those skilled in the art. When antigen-bindingactivity is not observed in the presence of a low concentration of thetarget tissue-specific compound, there is no dissociation and the valueof the upper limit becomes infinity.

For the value of antigen-binding activity, if the antigen is a solublemolecule, dissociation rate constant (kd) can be used; and if theantigen is a membrane-type molecule, apparent dissociation rate constant(apparent kd) can be used. The dissociation rate constant (kd) andapparent dissociation rate constant (apparent kd) can be determined bymethods known to those skilled in the art, for example, using Biacore(GE Healthcare), a flow cytometer, or such. In the present invention,when measuring the antigen-binding activity of an antigen-binding domain(or an antigen-binding molecule containing the domain) at a certainconcentration of the target tissue-specific compound, conditions otherthan the concentration of the compound concentration are preferably thesame.

For example, in an embodiment provided by the present invention, anantigen-binding domain (or an antigen-binding molecule containing thedomain) with lower antigen-binding activity in the absence of a targettissue-specific compound than in the presence of the compound, may beobtained by screening of antigen-binding domains (or antigen-bindingmolecules) that comprises the steps of:

(a) determining antigen-binding activity of antigen-binding domains (orantigen-binding molecules) in the absence of a target tissue-specificcompound;

(b) determining antigen-binding activity of the antigen-binding domains(or antigen-binding molecules) in the presence of the targettissue-specific compound; and

(c) selecting an antigen-binding domain (or an antigen-binding molecule)with lower antigen-binding activity in the absence of the targettissue-specific compound than in the presence of the compound.

For example, in an embodiment provided by the present invention, anantigen-binding domain (or an antigen-binding molecule containing thedomain) with lower antigen-binding activity in the presence of a lowconcentration of a target tissue-specific compound than in the presenceof a high concentration of the compound, may be obtained by screening ofantigen-binding domains (or antigen-binding molecules) that comprisesthe steps of:

(a) determining antigen-binding activity of antigen-binding domains (orantigen-binding molecules) in the presence of a low concentration of atarget tissue-specific compound;

(b) determining antigen-binding activity of the antigen-binding domains(or antigen-binding molecules) in the presence of a high concentrationof the target tissue-specific compound; and

(c) selecting an antigen-binding domain (or an antigen-binding molecule)with lower antigen-binding activity in the presence of a lowconcentration of the target tissue-specific compound than in thepresence of a high concentration of the compound.

Furthermore, in an embodiment provided by the present invention, anantigen-binding domain (or an antigen-binding molecule containing thedomain) with lower antigen-binding activity in the absence of a targettissue-specific compound than in the presence of the compound, may beobtained by screening of antigen-binding domains (or antigen-bindingmolecules) or a library thereof that comprises the steps of:

(a) contacting antigen-binding domains (or antigen-binding molecules) ora library thereof with an antigen in the presence of a targettissue-specific compound;

(b) placing antigen-binding domains (or antigen-binding molecules) thatbind to the antigen in said step (a) in the absence of the compound;

(c) isolating an antigen-binding domain (or an antigen-binding molecule)that dissociated in said step (b).

Furthermore, in an embodiment provided by the present invention, anantigen-binding domain (or an antigen-binding molecule containing thedomain) with lower antigen-binding activity in the presence of a lowconcentration of a target tissue-specific compound than in the presenceof a high concentration of the compound, may be obtained by screening ofantigen-binding domains (or antigen-binding molecules) or a librarythereof that comprises the steps of:

(a) contacting antigen-binding domains (or antigen-binding molecules) ora library thereof with an antigen in the presence of a highconcentration of a target tissue-specific compound;

(b) placing antigen-binding domains (or antigen-binding molecules) thatbind to the antigen in said step (a) in the presence of a lowconcentration of the compound;

(c) isolating an antigen-binding domain (or an antigen-binding molecule)that dissociates in said step (b).

Alternatively, in an embodiment provided by the present invention, anantigen-binding domain (or an antigen-binding molecule containing thedomain) with lower antigen-binding activity in the absence of a targettissue-specific compound than in the presence of the compound, may beobtained by screening of antigen-binding domains (or antigen-bindingmolecules) or a library thereof that comprises the steps of:

(a) contacting a library of antigen-binding domains (or antigen-bindingmolecules) with an antigen in the absence of a target tissue-specificcompound;

(b) selecting antigen-binding domains (or antigen-binding molecules)that do not bind to the antigen in said step (a);

(c) allowing the antigen-binding domains (or antigen-binding molecules)selected in said step (b) to bind to the antigen in the presence of thecompound; and

(d) isolating an antigen-binding domain (or an antigen-binding molecule)that binds to the antigen in said step (c).

Alternatively, in an embodiment provided by the present invention, anantigen-binding domain (or an antigen-binding molecule containing thedomain) with lower antigen-binding activity in the presence of a lowconcentration of a target tissue-specific compound than in the presenceof a high concentration of the compound, may be obtained by screening ofantigen-binding domains (or antigen-binding molecules) or a librarythereof that comprises the steps of:

(a) contacting a library of antigen-binding domains (or antigen-bindingmolecules) with an antigen in the presence of a low concentration of atarget tissue-specific compound;

(b) selecting antigen-binding domains (or antigen-binding molecules)that do not bind to the antigen in said step (a);

(c) allowing the antigen-binding domains (or antigen-binding molecules)selected in said step (b) to bind to the antigen in the presence of ahigh concentration the compound; and

(d) isolating an antigen-binding domain (or an antigen-binding molecule)that binds to the antigen in said step (c).

Furthermore, in an embodiment provided by the present invention, anantigen-binding domain (or an antigen-binding molecule containing thedomain) with lower antigen-binding activity in the absence of a targettissue-specific compound than in the presence of the compound, may beobtained by a screening method comprising the steps of:

(a) contacting a library of antigen-binding domains (or antigen-bindingmolecules) with an antigen-immobilized column in the presence of atarget tissue-specific compound;

(b) eluting an antigen-binding domain (or antigen-binding molecule) thatbinds to the column in said step (a) from the column in the absence ofthe compound; and

(c) isolating the antigen-binding domain (or antigen-binding molecule)eluted in said step (b). Furthermore, in an embodiment provided by thepresent invention, an antigen-binding domain (or an antigen-bindingmolecule containing the domain) with lower antigen-binding activity inthe presence of a low concentration of a target tissue-specific compoundthan in the presence of a high concentration of the compound, may beobtained by a screening method comprising the steps of:

(a) contacting a library of antigen-binding domains (or antigen-bindingmolecules) with an antigen-immobilized column in the presence of a highconcentration of a target tissue-specific compound;

(b) eluting an antigen-binding domain (or antigen-binding molecule) thatbinds to the column in said step (a) from the column in the presence ofa low concentration of the compound; and

(c) isolating the antigen-binding domain (or antigen-binding molecule)eluted in said step (b).

Furthermore, in an embodiment provided by the present invention, anantigen-binding domain (or an antigen-binding molecule containing thedomain) with lower antigen-binding activity in the absence of a targettissue-specific compound than in the presence of the compound, may beobtained by a screening method comprising the steps of:

(a) allowing a library of antigen-binding domains (or antigen-bindingmolecules) to pass through an antigen-immobilized column in the absenceof a target tissue-specific compound;

(b) collecting an antigen-binding domain (or antigen-binding molecule)eluted without binding to the column in said step (a);

(c) allowing the antigen-binding domain (or antigen-binding molecule)collected in said step (b) to bind to the antigen in the presence of thecompound; and

(d) isolating an antigen-binding domain (or antigen-binding molecule)that binds to the antigen in said step (c).

Furthermore, in an embodiment provided by the present invention, anantigen-binding domain (or an antigen-binding molecule containing thedomain) with lower antigen-binding activity in the presence of a lowconcentration of a target tissue-specific compound than in the presenceof a high concentration of the compound, may be obtained by a screeningmethod comprising the steps of:

(a) allowing a library of antigen-binding domains (or antigen-bindingmolecules) to pass through an antigen-immobilized column in the presenceof a low concentration of a target tissue-specific compound;

(b) collecting an antigen-binding domain (or antigen-binding molecule)eluted without binding to the column in said step (a);

(c) allowing the antigen-binding domain (or antigen-binding molecule)collected in said step (b) to bind to the antigen in the presence of ahigh concentration of the compound; and

(d) isolating an antigen-binding domain (or antigen-binding molecule)that binds to the antigen in said step (c).

Furthermore, in an embodiment provided by the present invention, anantigen-binding domain (or an antigen-binding molecule containing thedomain) with lower antigen-binding activity in the absence of a targettissue-specific compound than in the presence of the compound, may beobtained by a screening method comprising the steps of:

(a) contacting an antigen with a library of antigen-binding domains (orantigen-binding molecules) in the presence of a target tissue-specificcompound;(b) obtaining an antigen-binding domain (or antigen-binding molecule)that binds to the antigen in said step (a);(c) placing the antigen-binding domain (or antigen-binding molecule)obtained in said step (b) in the absence of the compound; and(d) isolating an antigen-binding domain (or antigen-binding molecule)whose antigen-binding activity in said step (c) is weaker than that ofthe reference selected in said step (b).

Furthermore, in an embodiment provided by the present invention, anantigen-binding domain (or an antigen-binding molecule containing thedomain) with lower antigen-binding activity in the presence of a lowconcentration of a target tissue-specific compound than in the presenceof a high concentration of the compound, may be obtained by a screeningmethod comprising the steps of:

(a) contacting an antigen with a library of antigen-binding domains (orantigen-binding molecules) in the presence of a high concentration of atarget tissue-specific compound;(b) obtaining an antigen-binding domain (or antigen-binding molecule)that binds to the antigen in said step (a);(c) placing the antigen-binding domain (or antigen-binding molecule)obtained in said step (b) in the presence of a low concentration of thecompound; and(d) isolating an antigen-binding domain (or antigen-binding molecule)whose antigen-binding activity in said step (c) is weaker than that ofthe reference selected in said step (b).

The above-mentioned steps may be repeated two or more times. Thus, thepresent invention provides an antigen-binding domain (or anantigen-binding molecule containing the domain) with lowerantigen-binding activity in the absence of a target tissue-specificcompound than in the presence of the compound, or an antigen-bindingdomain (or an antigen-binding molecule containing the domain) with lowerantigen-binding activity in the presence of a low concentration of atarget tissue-specific compound than in the presence of a highconcentration of the compound, obtained by screening methods thatfurther comprise the step of repeating steps (a) to (c) or (a) to (d)two or more times in the above-mentioned screening methods. The numberof repeats of steps (a) to (c) or (a) to (d) is not particularlylimited, and it is generally ten or less.

In the screening methods of the present invention, a targettissue-specific compound may be a compound defined by quantitativetarget tissue specificity such as presence in the target tissue at aconcentration (for example, high concentration or low concentration)different from the concentration in non-target tissues. For example, atarget tissue-specific compound is differentially present at anyconcentrations. However, generally, a target tissue-specific compoundcan be present at a concentration increased by at least 5%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 100%, at least 110%, at least120%, at least 130%, at least 140%, at least 150%, at least 2-fold, atleast 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, atleast 10³-fold, at least 10⁴-fold, at least 10⁵-fold, at least 10⁶-fold,or more, or up to infinity (when the compound is absent in non-targettissues).

The threshold differentiating low and high concentrations can be setappropriately according to the compound. For example, in a non-limitingembodiment of the threshold of ATP or adenosine, the threshold for alow-concentration condition may be selected appropriately from thevalues of 10 nM, 1 nM, 100 pM, 10 pM, 1 pM, and 0 M. Depending on thepredetermined threshold, the high-concentration condition may be setappropriately at a value selected from at least 110%, at least 120%, atleast 130%, at least 140%, at least 150%, at least twice, at leastfive-fold, at least 10-fold, at least 50-fold, at least 100-fold, atleast 10³-fold, at least 10⁴-fold, at least 10⁵-fold, and at least10⁶-fold the value of each threshold. Furthermore, in a non-limitingembodiment of PGE2, the threshold for a low-concentration condition maybe selected appropriately from the values of 10 pM, 1 pM, 100 fM, 10 fM,1 fM, and 0 M. Depending on the predetermined threshold, thehigh-concentration condition may be set appropriately at a valueselected from at least 110%, at least 120%, at least 130%, at least140%, at least 150%, at least twofold, at least five-fold, at least10-fold, at least 50-fold, at least 100-fold, at least 10³-fold, atleast 10⁴-fold, at least 10⁵-fold, and at least 10⁶-fold the value ofeach threshold. Furthermore, in a non-limiting embodiment of Kynurenine,the threshold for a low-concentration condition may be selectedappropriately from the values of 10 μM, 1 μM, 100 nM, 10 nM, and 1 nM,and 0 M. Depending on the predetermined threshold, thehigh-concentration condition may be set appropriately at a valueselected from at least 110%, at least 120%, at least 130%, at least140%, at least 150%, at least twofold, at least five-fold, at least10-fold, at least 50-fold, at least 100-fold, at least 10³-fold, atleast 10⁴-fold, at least 10⁵-fold, and at least 10⁶-fold the value ofeach threshold.

The antigen-binding activity of an antigen-binding domain (or anantigen-binding molecule) may be measured by a method known to thoseskilled in the art, and conditions other than the concentration of atarget tissue-specific compound can be set appropriately by one skilledin the art. The antigen-binding activity of an antigen-binding domain(or an antigen-binding molecule) can be assessed as dissociationconstant (KD), apparent dissociation constant (apparent KD),dissociation rate constant (kd), apparent dissociation rate constant(apparent kd), etc. They can be determined by methods known to thoseskilled in the art, for example, using Biacore (GE Healthcare), theScatchard plot, FACS, or such.

In the present invention, the step of selecting an antibody or anantigen-binding domain with higher antigen-binding activity in thepresence of a target tissue-specific compound than in the absence of thecompound has the same meaning as the step of selecting an antibody or anantigen-binding domain with lower antigen-binding activity in theabsence of a target tissue-specific compound than in the presence of thecompound.

In the present invention, the step of selecting an antibody or anantigen-binding domain with higher antigen-binding activity in thepresence of a high concentration of a target tissue-specific compoundthan in the presence of a low concentration of the compound has the samemeaning as the step of selecting an antibody or an antigen-bindingdomain with lower antigen-binding activity in the absence of a targettissue-specific compound than in the presence of the compound.

As long as antigen-binding activity in the absence of a targettissue-specific compound is lower than the antigen-binding activity inthe presence of the compound, the difference between antigen-bindingactivity in the presence of the compound and antigen-binding activity inthe absence of the compound is not particularly limited, but preferably,the antigen-binding activity in the presence of the compound relative tothe antigen-binding activity in the absence of the compound is twofoldor more, more preferably 10-fold or more, and even more preferably40-fold or more. The upper limit of the difference between theantigen-binding activities is not particularly limited, and as long asit can be produced by the techniques of those skilled in the art, anyvalue such as 400-fold, 1000-fold, or 10000-fold is possible. In theabsence of a target tissue-specific compound, when antigen-bindingactivity is not observed, this upper limit becomes infinity.

The antigen-binding domains (or antigen-binding molecules containing thedomains) of the present invention which are to be screened by theaforementioned screening methods may be any antigen-binding domains (orantigen-binding molecules); and for example, the above-mentionedantigen-binding domains (or antigen-binding molecules) can be screened.For example, antigen-binding domains (or antigen-binding molecules)having naturally-occurring sequences can be screened, andantigen-binding domains (or antigen-binding molecules) with substitutedamino acid sequences may be screened.

Library

According to one embodiment, the antigen-binding domain (or anantigen-binding molecule comprising this domain) of the presentinvention may be obtained from a library comprising mainly a pluralityof antigen-binding molecules having sequences different from oneanother, in which the antigen-binding domain comprises at least oneamino acid residue that changes the antigen-binding activity of theantigen-binding molecule depending on the concentration of a smallmolecule compound. A non-limiting embodiment of the small moleculecompound is, for example, a target tissue-specific compound or anunnatural compound. Examples of a target tissue-specific compoundinclude (1) primary metabolites of the Krebs cycle or the glycolyticpathway such as lactic acid, succinic acid, or citric acid, (2) aminoacids such as alanine, glutamic acid, or aspartic acid, (3) amino acidmetabolites such as kynurenine and metabolites thereof such asanthranilic acid, 3-hydroxykynurenine, and kynurenic acid, (4)arachidonic acid metabolites such as prostaglandin E2, and (5)nucleosides carrying a purine ring structure such as adenosine,adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosinemonophosphate (AMP). Below are examples of a library that comprisesmainly a plurality of such antigen-binding molecules having differentsequences from one another, in which the antigen-binding domaincomprises at least one amino acid residue that changes the bindingactivity of the antigen-binding molecule toward an antigen depending onadenosine and/or ATP which are target tissue-specific compounds.Libraries of antigen-binding molecules whose antigen-binding activityvaries depending on the concentration of a small molecule compound otherthan adenosine and/or ATP may also be applied appropriately according tothe examples described below.

Herein, a “library” refers to a set of a plurality of antigen-bindingmolecules or a plurality of fusion polypeptides comprisingantigen-binding molecules that have different sequences from oneanother, or nucleic acids or polynucleotides encoding these molecules orpolypeptides. Sequences of a plurality of antigen-binding molecules or aplurality of fusion polypeptides comprising antigen-binding molecules ina library are not uniform sequences, and the antigen-binding moleculesor fusion polypeptides comprising antigen-binding molecules havesequences that are different from one another.

Embodiments of the “library” in the present specification can providenot only libraries that can efficiently yield antigen-binding moleculeswhich bind to a target antigen in the presence of a small molecule butdo not bind to the target antigen in the absence of the small molecule(small-molecule dependence), but also libraries that can efficientlyyield antibodies which bind to a target antigen in the absence of asmall molecule and do not bind to the target antigen in the presence ofthe small molecule (inverse small-molecule dependence).

In one embodiment of the present invention, a fusion polypeptide of theantigen-binding molecule of the present invention and a heterologouspolypeptide can be prepared. In a certain embodiment, the fusionpolypeptide can be formed by fusion with at least a portion of a viralcoat protein selected from the group consisting of, for example, viralcoat proteins pIII, pVIII, pVII, pIX, Soc, Hoc, gpD, and pVI, andmutants thereof.

In one embodiment, the antigen-binding molecule of the present inventionmay be ScFv, a Fab fragment, F(ab)₂, or F(ab′)₂. Therefore, in anotherembodiment, the present invention provides a library that comprisesmainly a plurality of fusion polypeptides having different sequencesfrom one another, in which the fusion polypeptides are formed by fusingthese antigen-binding molecules with a heterologous polypeptide.Specifically, the present invention provides a library that comprisesmainly a plurality of fusion polypeptides having different sequencesfrom one another, in which the fusion polypeptides are formed by fusingthese antigen-binding molecules with at least a portion of a viral coatprotein selected from the group consisting of, for example, viral coatproteins pIII, pVIII, pVII, pIX, Soc, Hoc, gpD, and pVI, and mutantsthereof. The antigen-binding molecule of the present invention mayfurther comprise a dimerization domain. In one embodiment, thedimerization domain can be located between the heavy or light chainvariable region of the antibody and at least a portion of the viral coatprotein. This dimerization domain may comprise at least one dimerizationsequence and/or one or more sequences comprising cysteine residue(s).This dimerization domain may be preferably linked to the C terminus ofthe heavy chain variable region or constant region. The dimerizationdomain can assume various structures, depending on whether the antibodyvariable region is prepared as a fusion polypeptide component with theviral coat protein component (an amber stop codon following thedimerization domain is absent) or depending on whether the antibodyvariable region is prepared predominantly without containing the viralcoat protein component (e.g., an amber stop codon following thedimerization domain is present). When the antibody variable region isprepared predominantly as a fusion polypeptide with the viral coatprotein component, bivalent display is achieved by one or more disulfidebonds and/or a single dimerization sequence.

Herein, the phrase “sequences are different from one another” in theexpression “a plurality of antigen-binding molecules whose sequences aredifferent from one another” means that the sequences of antigen-bindingmolecules in a library are different from one another. Specifically, ina library, the number of sequences different from one another reflectsthe number of independent clones with different sequences, and may alsobe referred to as “library size”. The library size of a conventionalphage display library ranges from 10⁶ to 10¹². The library size can beincreased up to 10¹⁴ by the use of known techniques such as ribosomedisplay. However, the actual number of phage particles used in panningselection of a phage library is in general 10 to 10,000 times greaterthan the library size. This excess multiplicity is also referred to as“the number of library equivalents”, and means that there are 10 to10,000 individual clones that have the same amino acid sequence. Thus,in the present invention, the phrase “sequences are different from oneanother” means that the sequences of independent antigen-bindingmolecules in a library, excluding library equivalents, are differentfrom one another. More specifically, the above means that there are 10⁶to 10¹⁴ antigen-binding molecules whose sequences are different from oneanother, preferably 10⁷ to 10¹² molecules, more preferably 10⁸ to 10¹¹molecules, and particularly preferably 10⁸ to 10¹⁰ molecules whosesequences are different from one another.

Herein, the phrase “a plurality of” in the expression “a library mainlycomposed of a plurality of antigen-binding domains or antigen-bindingmolecules containing an antigen-binding domain” generally refers to, inthe case of, for example, antigen-binding molecules, fusionpolypeptides, polynucleotide molecules, vectors, or viruses of thepresent invention, a group of two or more types of the substance. Forexample, when two or more substances are different from one another in aparticular characteristic, this means that there are two or more typesof the substance. Such examples may include, for example, mutant aminoacids observed at specific amino acid positions in an amino acidsequence. For example, when there are two or more antigen-bindingmolecules of the present invention whose sequences are substantially thesame or preferably the same except for flexible residues or except forparticular mutant amino acids at hypervariable positions exposed on thesurface, there are a plurality of antigen-binding molecules of thepresent invention. In another example, when there are two or morepolynucleotide molecules whose sequences are substantially the same orpreferably the same except for nucleotides encoding flexible residues ornucleotides encoding mutant amino acids of hypervariable positionsexposed on the surface, there are a plurality of polynucleotidemolecules of the present invention.

In addition, herein, the phrase “mainly composed of” in the expression“a library mainly composed of a plurality of antigen-binding molecules”reflects the number of antigen-binding molecules whose antigen-bindingactivity varies depending on the concentration of a small moleculecompound (e.g., a target tissue-specific compound), among independentclones with different sequences in a library. Specifically, it ispreferable that there are at least 10⁴ antigen-binding molecules havingsuch binding activity in a library. More preferably, antigen-bindingdomains of the present invention can be obtained from a librarycontaining at least 10⁵ antigen-binding molecules having such bindingactivity. Still more preferably, antigen-binding domains of the presentinvention can be obtained from a library containing at least 10⁶antigen-binding molecules having such binding activity. Particularlypreferably, antigen-binding domains of the present invention can beobtained from a library containing at least 10⁷ antigen-bindingmolecules having such binding activity. Yet more preferably,antigen-binding domains of the present invention can be obtained from alibrary containing at least 10⁸ antigen-binding molecules having suchbinding activity. Alternatively, this may also be preferably expressedas the ratio of the number of antigen-binding molecules in whichantigen-binding activity of the antigen-binding domain varies dependingon the presence or absence of adenosine and/or ATP with respect to thenumber of independent clones having different sequences in a library.Specifically, antigen-binding domains of the present invention can beobtained from a library in which antigen-binding molecules having suchbinding activity account for 10⁻⁶% to 80%, preferably 10⁻⁵% to 60%, morepreferably 10⁻⁴% to 40% of independent clones with different sequencesin the library. In the case of fusion polypeptides, polynucleotidemolecules, or vectors, similar expressions may be possible using thenumber of molecules or the ratio to the total number of molecules. Inthe case of viruses, similar expressions may also be possible using thenumber of virions or the ratio to total number of virions. As anon-limiting embodiment of the present invention, when a plurality ofantigen-binding molecules bind to a single type of antigen, preferably,at least 10, 100, 1000, 10⁴, 10⁵, 10⁶, 10⁷, or 10⁸ molecules are presentin a library of antigen-binding molecules showing such binding activity.More preferably, antigen-binding domains of the present invention may beobtained from a library in which at least ten antigen-binding moleculesshowing such binding activity are present. More preferably, theantigen-binding domains of the present invention may be obtained from alibrary in which at least 100 antigen-binding molecules showing suchbinding activity are present. Particularly preferably, theantigen-binding domains of the present invention may be obtained from alibrary in which at least 1000 antigen-binding molecules showing suchbinding activity are present.

An embodiment of the present invention provides a library produced by amethod that comprises the steps of:

(a) identifying amino acid sites that fulfill any one or more of (i) to(iii) below in antigen-binding domains whose antigen-binding activityvaries depending on the concentration of a small molecule compound or inantigen-binding domains that have binding activity to a small moleculecompound:

(i) one or more amino acid sites that are not involved in the binding tothe small molecule compound;

(ii) one or more amino acid sites that show diversity of amino acidoccurrence frequency in the antibody repertoire of the animal species towhich the parent antigen-binding domain belongs; and

(iii) one or more amino acid sites that are not important for canonicalstructure formation; and

(b) designing a library that comprises nucleic acids encoding unmodifiedantigen-binding domains/molecules, and nucleic acids that encodeindividually a plurality of variants of the aforementionedantigen-binding domains or antigen-binding molecules comprising anantigen-binding domain, which have different sequences from one anotherand have modifications at one or more of the amino acid sites identifiedin step (a).

In the present invention, “one or more amino acid sites that are notinvolved in the binding to the small molecule compound” can beidentified by methods such as crystal structure analysis of a complexformed by a small molecule compound and an antibody, three-dimensionalstructure analysis using NMR, or introduction of amino acid mutations.In a non-limiting embodiment of the present invention, antibody residuesthat are not involved in the binding to the small molecule can beidentified from crystal structure analysis of the complex formed by thesmall molecule and the antibody. The phrase “involved in the binding tothe small molecule” as used herein refers to a condition whereintermolecular interactions are taking place between the atoms of themain chain or side chains of the amino acids forming the antibody Hchain or L chain and the atoms of the small molecule compound at adistance that may have an effect on the binding activity; or a conditionwhere certain amino acid residues are involved in the binding of thesmall molecule compound, including an indirect effect of stabilizing thethree-dimensional structure of the CDR loop and such to the conformationwhen bound to the small molecule compound; and a condition thatsatisfies both of those conditions.

The “condition where intermolecular interactions are taking place” inthe present specification can be determined based on the interatomicdistances, for example, between non-hydrogen atoms constituting the mainchain or side chains of the amino acids that form the antibody H chainor L chain and the non-hydrogen atoms constituting the small moleculecompound obtained from crystal structure analysis of the complex formedby the small molecule and the antibody. For example, the above-mentionedinteratomic distances are preferably 3.0 Å, 3.2 Å, 3.4 Å, 3.6 Å, 3.8 Å,4.0 Å, 4.2 Å, 4.4 Å, 4.6 Å, 4.8 Å, or 5.0 Å or less, but are not limitedthereto. More preferably, examples of the interatomic distance are 3.6Å, 3.8 Å, 4.0 Å, or 4.2 Å or less.

More specifically, the possibility of a direct interaction can bedetermined based on information on the interatomic distances in thethree-dimensional structure and the types of intermolecular interactionsthat take place, and information on the types of atoms. Thedetermination can be done with more accuracy by, without being limitedthereto, observing the effect of introducing amino acid residuemutations such as modification to Ala or Gly on the activities of smallmolecule compounds.

With respect to the “indirectly influenced condition” in the presentspecification, whether there is an indirect effect on the binding to asmall molecule can be estimated, for example, by analyzing in detailconditions of the conformation of each amino acid residue andintermolecular interactions with the surrounding residues from thethree-dimensional structure of the small-molecule-antibody complex. Thedetermination can be done more accurately by observing the effect ofintroducing amino acid residue mutations such as modification to Ala orGly on the activities of small molecule compounds.

In one embodiment of the present invention, one can select amino acidsthat are capable of maintaining an appropriate level of binding to thecompound, even when residues that are identified not to be involved insmall-molecule-binding are substituted with those amino acids.Accordingly, one can design a library in which selected amino acidsappear at the selected residues. In this case, one can design a libraryto comprise mainly a plurality of antigen-binding molecules, which is anassembly of antigen-binding molecules whose residues identified to benot involved in binding of the small molecule compound have beensubstituted with amino acids that are different from one another.

In another embodiment, amino acid sites that are not involved in bindingto a small molecule compound can be considered as amino acid sites otherthan any one or more amino acid sites selected from among the amino acidsites involved in binding to a small molecule compound.

In a non-limiting embodiment of the present invention, “one or moreamino acid sites not involved in binding to a small molecule compound”can be identified by methods of introducing amino acid mutations. Forexample, amino acids of the variable region are comprehensivelymodified, and the binding of each variant to the small molecule ismeasured by known methods that use Biacore and such. The bindingactivity (affinity) of each variant to the small molecule is calculatedas a KD value. This KD value is compared with the KD value of anunmodified antigen-binding domain/molecule which is the parent sequence,and the modified positions that show binding greater than a certainstandard are determined as amino acid sites not involved in binding tothe small molecule compound. For example, as a result of performingmeasurements using known methods such as Biacore, the binding activity(affinity) of the individual variants to the small molecule iscalculated as a KD value; and sites of the heavy chain where alterationdoes not reduce the binding capacity to the small molecule to less than1/100, 1/50, 1/10, 1/9, ⅛, 1/7, ⅙, ⅕, ¼, ⅓, or ½ of the unmodifiedantigen-binding domain/molecule, and sites of the light chain wherealteration does not reduce the binding capacity to the small molecule toless than 1/100, 1/50, 1/10, 1/9, ⅛, 1/7, ⅙, ⅕, ¼, ⅓, or ½ of theunmodified antigen-binding domain/molecule are determined as amino acidsites not involved in binding to the small molecule compound, but theabove-mentioned standards are non-limiting. Alternatively, instead ofcomparing with the KD value of the unmodified antigen-bindingdomain/molecule which is the parent sequence, the binding activity(affinity) of individual variants to the small molecule is calculated asa KD value, and heavy chain sites having binding capacity not lower than10 mM, 1 mM, 100 uM, 10 uM, 1 uM, 100 nM, 10 nM, 1 nM, 100 pM, 10 pM, or1 pM, and light chain sites having binding capacity not lower than 10mM, 1 mM, 100 uM, 10 uM, 1 uM, 100 nM, 10 nM, 1 nM, 100 pM, 10 pM, or 1pM are determined as amino acid sites not involved in binding to thesmall molecule compound, but the above-mentioned standards arenon-limiting. The binding activity of the unmodified antigen-bindingdomain/molecule and variants to the small molecule can be measured byappropriately selecting methods known to those skilled in the art(Biacore, ELISA, ECL, and such).

In another embodiment, amino acid sites that are not involved in bindingto a small molecule compound can be considered as amino acid sites otherthan any one or more amino acid sites selected from among the amino acidsites involved in binding to the small molecule compound.

“Designing a library comprising nucleic acids that encode individually aplurality of variants of the aforementioned antigen-binding domains orantigen-binding molecules comprising an antigen-binding domain, whichhave different sequences from one another” in the present inventionincludes designing a library that comprises a plurality of variants ofantigen-binding domains or antigen-binding molecules comprising anantigen-binding domain whose amino acids at specified sites have beenmodified to desired amino acids using known library techniques such asNNK and TRIM libraries (Gonzalez-Munoz A et al. MAbs 2012; Lee C V etal. J Mol Biol. 2004; Knappik A. et al. J Mol Biol. 2000; Tiller T etal. MAbs 2013), but is not particularly limited to this embodiment.

“One or more amino acids” in the present invention does not particularlylimit the number of amino acids, and may be two or more types of aminoacids, five or more types of amino acids, ten or more types of aminoacids, 15 or more types of amino acids, or 20 or more types of aminoacids.

“Amino acid sites showing diversity of amino acid occurrence frequency”in the present invention refers to amino acid sites where two or moretypes of amino acids are found to be present at an occurrence frequencyof 1% or higher in the antibody repertoire of the animal species towhich the parent antibody (parent antigen-binding domain) belongs.

“Parent antigen-binding domain” in the present invention refers to anantigen-binding domain whose antigen-binding activity varies dependingon the concentration of a small molecule compound or an antigen-bindingdomain having binding activity to a small molecule compound, which willserve as a template for library production.

“Antibody repertoire of the animal species to which the parentantigen-binding domain belongs” in the present invention refers to arepertoire of antibody gene sequences found in the genes of animalspecies from which the corresponding parent antigen-binding domain isderived. Without being limited thereto, as an example, when thecorresponding parent antigen-binding domain is derived from a human, theantibody repertoire of the animal species to which the parentantigen-binding domain belongs refers to a repertoire of antibody genesequences found in human genes, and when the corresponding parentantigen-binding domain is derived from a rabbit, the antibody repertoireof the animal species to which the parent antigen-binding domain belongsrefers to a repertoire of antibody gene sequences found in the genes ofrabbits. However, it must be noted that sequences that are actually notexpressed as antibodies due to frame shift or presence oftermination/initiation codons are not included even if they are presentin the genes.

When the corresponding parent antigen-binding domain is derived from anon-human animal, it can be humanized according to conventional methods,and such techniques are widely known to those skilled in the art (forexample, European patent publication EP239400, internationalpublications WO1996/002576, WO1993/012227, WO1992/003918, WO1994/002602,WO1994/025585, WO1996/034096, WO1996/033735, WO1992/001047,

WO1992/020791, WO1993/006213, WO1993/011236, WO1993/019172,WO1995/001438, and WO1995/015388, Cancer Res., (1993) 53, 851-856, andBBRC., (2013) 436(3):543-50). When a corresponding parentantigen-binding domain is humanized according to conventional methodsand then made into a library, in the “antibody repertoire of the animalspecies to which the parent antigen-binding domain belongs” of thepresent invention, the antigen-binding domain prior to humanization andthe humanized antigen-binding domain can be both treated as the parentantigen-binding domain. Accordingly, the human repertoire and therepertoire of the animal species from which the pre-humanizationantigen-binding domains are derived can be both applied as a repertoireof the same animal species. Without being limited thereto, as anexample, when the antigen-binding domains prior to humanization arederived from rabbits, the antibody repertoire of the animal species towhich the parent antigen-binding domain belongs refers to the repertoireof antibody gene sequences found in the genes of humans and/or rabbits.However, it must be noted that sequences that are not actually expressedas antibodies due to frame shift or presence of termination/initiationcodons are not included even if they are present in the genes.

As an example, the antibody repertoire of the animal species to whichthe parent antigen-binding domain belongs can be investigated byreferring to a known database, without being limited thereto. The sitewhere there is diversity of the amino acid occurrence frequency isgenerally in the CDR region. In one embodiment, when determining thehypervariable positions of known and/or naturally-occurring antibodies,the data provided by Kabat, Sequences of Proteins of ImmunologicalInterest (National Institute of Health Bethesda Md., 1987 and 1991) areuseful. Furthermore, multiple databases on the Internet(http://vbase.mrc-cpe.cam.ac.uk/ and http://www.bioinforg.uk/abs/index.html) provide many collected sequences of human lightchains and heavy chains, and their locations. Information on thesequences and their locations is useful for determining thehypervariable positions in the present invention.

In another embodiment, the antibody repertoire of the animal species towhich the parent antigen-binding domain belongs can be examined bycloning antibody genes obtained from the corresponding animal speciesand analyzing their sequences. Without being limited thereto, as anexample, a human antibody repertoire is constructed from antibody genesderived from lymphocytes of healthy individuals and may be examined byanalyzing the sequences of a naive library comprising naive sequenceswhich are unbiased antibody sequences in their repertoire (Gejima et al.(Human Antibodies (2002) 11, 121-129); Cardoso et al. (Scand. J.Immunol. (2000) 51, 337-344)). When examining a repertoire, it isdesirable to analyze at least 100 types of sequences, preferably 200types of sequences, and more preferably 400 types of sequences or more.

With respect to “the antibody repertoire of the animal species to whichthe parent antigen-binding domain belongs” in the present invention,more preferably it is desirable to examine subgroups of the germline towhich the parent antigen-binding domain belongs, without being limitedthereto. Examples of a framework include sequences of currently knowncompletely human-type framework regions listed in a website such asV-Base (http://vbase.mrc-cpe.cam.ac.uk/). Any of the sequences of theseframework regions may be appropriately used as a germline sequencecontained in the antigen-binding molecule of the present invention. Thegermline sequences may be classified into subgroups based on theirsimilarity (Tomlinson et al., J. Mol. Biol. (1992) 227, 776-798;Williams and Winter, Eur. J. Immunol. (1993) 23, 1456-1461; and Cox etal., Nat. Genetics (1994) 7, 162-168). In one example, seven subgroupsfor the heavy-chain variable region in human antibodies, seven subgroupsfor Vκ, and ten types of subgroups for Vλ have been reported; andwithout being particularly limited to this embodiment, each of the aminoacid sites may be examined by analyzing the amino acid repertoire in thesubgroup to which the parent antigen-binding domain belongs.

In the “amino acid sites that are not important for canonical structureformation” of the present invention, an antibody canonical structureshows clustering of the three-dimensional structures of mainly CDR1 andCDR2 of the antibody heavy chains and light chains, and the structurescan be classified according to the antibody subgroups and the length orsequence of CDRs. In each canonical structure, residues important formaintaining the structure are already known, and by referring to thereports of Chothia et al. (J. Mol. Biol. (1992) 227, 799-817),Al-Lazikani et al. (J. Mol. Biol. (1997) 273, 927-948), Tomlinson et al.(J. Mol. Biol. (1992) 227, 776-798) and such, it is possible to identifythe canonical structure that the corresponding parent antigen-bindingmolecule is classified to, and the residues important for thatstructure.

Furthermore, even in antigen-binding domains other than those ofantibodies, it is known that there are residues important formaintaining the structure; and while not being limited thereto, aminoacid sites not important for formation of the canonical structure ineach antigen-binding domain can be identified by structural analysis andsuch of produced mutants.

Another embodiment of the library of the present invention is, forexample, the library below.

A library which is produced by a method comprising the steps of:

(a) identifying amino acid sites that fulfill any one or more of (i) to(iii) below in antigen-binding domains whose antigen-binding activityvaries depending on the concentration of a small molecule compound or inantigen-binding domains that have binding activity to a small moleculecompound:

(i) one or more amino acid sites that are not involved in the binding tothe small molecule compound;

(ii) one or more amino acid sites that show diversity of amino acidoccurrence frequency in the antibody repertoire of the animal species towhich the parent antigen-binding domain belongs; and

(iii) one or more amino acid sites that are not important for canonicalstructure formation;

(b) producing a plurality of variants of the aforementionedantigen-binding domains or antigen-binding molecules comprising anantigen-binding domain, which have different sequences from one anotherand have modifications at one or more of the amino acid sites identifiedin step (a);

(c) identifying one or more amino acid modifications that do notsubstantially change the binding activity of each of the aforementionedvariants to the small molecule compound; and

(d) producing a library comprising nucleic acids that encode unmodifiedantigen-binding domains/molecules, and nucleic acids that encode aplurality of variants of the aforementioned antigen-binding domains orantigen-binding molecules comprising the antigen-binding domain, whichhave different sequences from one another and have one or more of theamino acid modifications identified in step (c).

In the “step of producing a plurality of variants of the aforementionedantigen-binding domains or antigen-binding molecules comprising anantigen-binding domain, which have different sequences from one another”of the present invention, among the amino acid sites identified in step(a), the sites in CDR1 and CDR2 can be substituted with amino acidshaving an occurrence frequency of 10% or more, 9% or more, 8% or more,7% or more, 6% or more, 5% or more, 4% or more, 3% or more, 2% or more,or 1% or more in the germline, and the sites in CDR3 can be substitutedwith amino acids having an occurrence frequency of 10% or more, 9% ormore, 8% or more, 7% or more, 6% or more, 5% or more, 4% or more, 3% ormore, 2% or more, or 1% or more in the germline to produce theindividual variants, but the production is not limited thereto.

“Plurality of variants” in the present invention refers to individuallydifferent variants of antigen-binding domains produced by substitutingat least one or more amino acids in the unmodified antigen-bindingdomain which is the parent sequence.

The “step of identifying one or more amino acid modifications that donot substantially change the binding activity of each of theaforementioned variants to the small molecule compound” in the presentinvention has the meaning below. For example, the binding of eachvariant to a small molecule is measured by a known method using Biacoreor such, and the binding activity (affinity) of each variant to thesmall molecule is calculated as a KD value. This KD value is compared tothe KD value of the unmodified antigen-binding domain/molecule which isthe parent sequence, and the modified positions that show bindinggreater than a certain standard are determined as sites that can bechanged; and without being limited thereto, amino acids substituted atthese sites can be determined as amino acids that can be made into alibrary (flexible residues made to appear in the library).Alternatively, rather than comparing the KD value of an individualvariant with the KD value of the unmodified antigen-bindingdomain/molecule which is the parent sequence, one can determine themodified positions that show binding greater than a certain standard assites that can be changed; and without being limited thereto, aminoacids substituted at these sites can be determined as amino acids thatcan be made into a library (flexible residues made to appear in thelibrary).

In the present invention, in determining amino acids that can be madeinto a library, “modified positions that show binding greater than acertain standard” has the meaning below. For example, as a result ofperforming measurements using known methods such as Biacore, the bindingactivity (affinity) of each variant to a small molecule is calculated asa KD value, and sites of the heavy chain where alteration does notreduce the binding capacity to the small molecule to less than 1/100,1/50, 1/10, 1/9, ⅛, 1/7, ⅙, ⅕, ¼, ⅓, or ½ of the unmodifiedantigen-binding domain/molecule, and sites of the light chain wherealteration does not reduce the binding capacity to the small molecule toless than 1/100, 1/50, 1/10, 1/9, ⅛, 1/7, ⅙, ⅕, ¼, ⅓, or ½ of theunmodified antigen-binding domain/molecule are determined as sites thatcan be changed; and amino acids substituted at these sites can bedetermined as amino acid sites that can be made into a library, but theabove-mentioned standards are non-limiting. Alternatively, rather thancomparing with the KD value of the unmodified antigen-bindingdomain/molecule which is the parent sequence, the binding activity(affinity) of an individual variant to a small molecule is calculated asa KD value, and heavy chain sites having binding capacity not lower than10 mM, 1 mM, 100 uM, 10 uM, 1 uM, 100 nM, 10 nM, 1 nM, 100 pM, 10 pM, or1 pM, and light chain sites having binding capacity not lower than 10mM, 1 mM, 100 uM, 10 uM, 1 uM, 100 nM, 10 nM, 1 nM, 100 pM, 10 pM, or 1pM are determined as sites that can be changed; and amino acidssubstituted at these sites can be determined as amino acid positionsthat can be made into a library, but the above-mentioned standards arenon-limiting.

The binding activity of the unmodified antigen-binding domain/moleculeand variants to a small molecule can be measured by appropriatelyselecting methods known to those skilled in the art (Biacore, ELISA,ECL, and such).

In another embodiment, amino acid sites not involved in binding to asmall molecule compound may be considered as amino acid sites other thanany one or more amino acid sites selected from among the amino acidsites involved in binding to a small molecule compound.

In the present invention, “the step of producing a library comprisingnucleic acids that encode unmodified antigen-binding domains/molecules,and a plurality of variants of the aforementioned antigen-bindingdomains or antigen-binding molecules comprising an antigen-bindingdomain, which have different sequences from one another and have one ormore of the amino acid modifications identified in step (d)” includesbut is not limited to embodiments of constructing a library so that theoccurrence frequency of each amino acid identified in step (d) atspecific site will become equivalent (for example, when the amino acidrepertoire is ten, the occurrence of each amino acid will be adjusted to10%).

Another embodiment of the library of the present invention is, forexample, the library below.

A library produced by a method that comprises the steps of:

1) contacting a library that comprises a plurality of antigen-bindingmolecules having binding activity to a small molecule compound with thesmall molecule compound; and

2) concentrating from the library, nucleic acids that encode a pluralityof variants of antigen-binding molecules having binding activity to thesmall molecule compound.

Furthermore, in another embodiment, the library is a library in whichthe antigen-binding molecule is an antigen-binding molecule comprisingthe heavy-chain variable regions and light-chain variable regions of anantibody, and is produced by a method that comprises any one of thesteps of:

1) designing a library by concentrating nucleic acids that encode aplurality of variants of antigen-binding molecules having bindingactivity to a small molecule compound from the library which comprisesnucleic acids encoding one or more variants produced by modifying aminoacids positioned in the heavy chain variable regions;

2) designing a library by concentrating nucleic acids that encode aplurality of variants of antigen-binding molecules having bindingactivity to a small molecule compound from the library which comprisesnucleic acids encoding one or more variants produced by modifying aminoacids positioned in the light chain variable regions; and

3) designing a library by combining the antigen-bindingmolecule-encoding nucleic acids concentrated from each of the variableregion libraries of steps 1) and 2).

“Concentrate” in the present invention refers to increasing the ratio ofnucleic acids encoding variants having the desired activity present inthe library relative to the ratio in the library before theconcentration operation is performed. Without being limited thereto, asan example, concentrating nucleic acids that encode variants ofantigen-binding molecules having binding activity to a small moleculecompound can be accomplished by increasing the ratio of presence ofnucleic acids encoding the variants of antigen-binding molecules havingbinding activity to the small molecule compound by panning. Morespecifically, without being limited thereto, as an example, it ispossible to increase the ratio of presence of nucleic acids that encodevariants of antigen-binding molecules having binding activity to a smallmolecule compound by panning, which involves contacting the smallmolecule compound with phages presenting a library that comprises aplurality of antigen-binding molecules on their surface by the phagedisplay method, removing phages presenting molecules that do not havebinding activity and phages not presenting the molecules by a washingoperation, and then collecting only the phages that presentantigen-binding molecules which maintain binding. More specifically, theratio of presence of nucleic acids that encode variants having thedesired activity increases preferably 1.1-times or more relative to thatof the library before the concentration operation is performed. Morepreferably, the library of the present invention can be produced byincreasing the ratio of presence of nucleic acids encoding variantshaving the desired activity by 1.2 times or more, 1.5 times or more, 2times or more, 4 times or more, 10 times or more, 25 times or more, or100 times or more.

Library Production Method

The invention of the present application also relates to methods forproducing various embodiments of “libraries” included in the inventionof this application described above.

The “library production method” of the invention of the presentapplication is not limited to any of the specific methods shown asexamples below, and includes any method that can produce theabove-described “libraries” of the invention of the present application.

For example, “library production method” in the invention of the presentapplication include the methods shown as examples below.

Each of the specific matters in the “library production method” shown asexamples below has technical significance as described in detail abovewith regard to the “library” in the invention of the presentapplication.

Example 1

A method for producing a library that comprises mainly

(i) a plurality of antigen-binding domains or antigen-binding moleculescomprising an antigen-binding domain, which have different sequencesfrom one another; or

(ii) nucleic acids that encode the plurality of antigen-binding domainsor antigen-binding molecules comprising an antigen-binding domain, whichhave different sequences from one another;

wherein the aforementioned antigen-binding molecule domains orantigen-binding molecules are antigen-binding domains or antigen-bindingmolecules comprising an antigen-binding domain whose antigen-bindingactivity varies depending on the concentration of a small moleculecompound;wherein the method comprises the steps of (a) and (b) below:

(a) identifying amino acid sites that fulfill any one or more of (i) to(iii) below in antigen-binding domains whose antigen-binding activityvaries depending on the concentration of a small molecule compound or inantigen-binding domains that have binding activity to a small moleculecompound:

(i) one or more amino acid sites that are not involved in the binding tothe small molecule compound;

(ii) one or more amino acid sites that show diversity of amino acidoccurrence frequency in the antibody repertoire of the animal species towhich the parent antigen-binding domain belongs; and

(iii) one or more amino acid sites that are not important for canonicalstructure formation; and (b) designing a library comprising nucleicacids that encode unmodified antigen-binding domains/molecules, andnucleic acids that encode individually a plurality of variants of theaforementioned antigen-binding domains or antigen-binding moleculescomprising an antigen-binding domain, which have different sequencesfrom one another and have modifications at one or more of the amino acidsites identified in step (a).

Example 2

A method for producing a library that comprises mainly

(i) a plurality of antigen-binding domains or antigen-binding moleculescomprising an antigen-binding domain, which have different sequencesfrom one another; or

(ii) nucleic acids that encode the plurality of antigen-binding domainsor antigen-binding molecules comprising an antigen-binding domain, whichhave different sequences from one another;

wherein the aforementioned antigen-binding molecule domains orantigen-binding molecules are antigen-binding domains or antigen-bindingmolecules comprising an antigen-binding domain whose antigen-bindingactivity varies depending on the concentration of a small moleculecompound;wherein the method comprises the steps of (a) to (d) below:

(a) identifying amino acid sites that fulfill any one or more of (i) to(iii) below in antigen-binding domains whose antigen-binding activityvaries depending on the concentration of a small molecule compound or inantigen-binding domains that have binding activity to a small moleculecompound:

(i) one or more amino acid sites that are not involved in the binding tothe small molecule compound;

(ii) one or more amino acid sites that show diversity of amino acidoccurrence frequency in the antibody repertoire of the animal species towhich the parent antigen-binding domain belongs; and

(iii) one or more amino acid sites that are not important for canonicalstructure formation;

(b) producing a plurality of variants of the aforementionedantigen-binding domains or antigen-binding molecules comprising anantigen-binding domain, which have different sequences from one anotherand have modifications at one or more of the amino acid sites identifiedin step (a);

(c) identifying one or more amino acid modifications that do notsubstantially change the binding activity of each of the aforementionedvariants to the small molecule compound; and

(d) producing a library comprising nucleic acids that encode unmodifiedantigen-binding domains/molecules, and nucleic acids that encode aplurality of variants of the aforementioned antigen-binding domains orantigen-binding molecules comprising an antigen-binding domain, whichhave different sequences from one another and have one or more of theamino acid modifications identified in step (c).

Example 3

A method for producing a library that comprises mainly

(i) a plurality of antigen-binding domains or antigen-binding moleculescomprising an antigen-binding domain, which have different sequencesfrom one another; or

(ii) nucleic acids that encode the plurality of antigen-binding domainsor antigen-binding molecules comprising an antigen-binding domain, whichhave different sequences from one another;

wherein the aforementioned antigen-binding molecule domains orantigen-binding molecules are antigen-binding domains or antigen-bindingmolecules comprising an antigen-binding domain whose antigen-bindingactivity varies depending on the concentration of a small moleculecompound;wherein the method comprises the steps of 1) and 2) below:

1) contacting a library that comprises a plurality of antigen-bindingmolecules having binding activity to a small molecule compound with thesmall molecule compound; and

2) concentrating from the library, nucleic acids that encode a pluralityof variants of antigen-binding molecules having binding activity to thesmall molecule compound.

Example 4

A method for producing a library that comprises mainly

(i) a plurality of antigen-binding domains or antigen-binding moleculescomprising an antigen-binding domain, which have different sequencesfrom one another; or

(ii) nucleic acids that encode the plurality of antigen-binding domainsor antigen-binding molecules comprising an antigen-binding domain, whichhave different sequences from one another;

wherein the aforementioned antigen-binding molecule domains orantigen-binding molecules are antigen-binding domains or antigen-bindingmolecules comprising an antigen-binding domain whose antigen-bindingactivity varies depending on the concentration of a small moleculecompound;wherein the method comprises any one of steps 1) to 3) below:

1) designing a library by concentrating nucleic acids that encode aplurality of variants of antigen-binding molecules having bindingactivity to a small molecule compound from the library of (Example 3)which comprises nucleic acids encoding one or more variants produced bymodifying amino acids positioned in the heavy chain variable regions;

2) designing a library by concentrating nucleic acids that encode aplurality of variants of antigen-binding molecules having bindingactivity to a small molecule compound from the library of (Example 3)which comprises nucleic acids encoding one or more variants produced bymodifying amino acids positioned in the light chain variable regions;and

3) designing a library by combining the antigen-bindingmolecule-encoding nucleic acids concentrated from each of the variableregion libraries of steps 1) and 2).

Example 5

The library production method of any one of (Example 1) to (Example 4)described above, wherein the antigen-binding molecules are fusionpolypeptides formed by fusing an antigen-binding domain with at least aportion of a virus coat protein.

Example 6

The library production method of any one of (Example 1) to (Example 4)described above, wherein the aforementioned antigen-binding moleculesare antigen-binding molecules comprising antibody heavy chains and lightchains, and the method further comprises the step of designing asynthetic library of the heavy chains and/or light chains.

Example 7

The library production method of (Example 6) described above, whereinthe antibody heavy chains and/or light chains include a germline-derivedframework sequence.

Example 8

The library production method of any one of (Example 1) to (Example 7)described above, wherein the aforementioned small molecule compound is atarget tissue-specific compound or an unnatural compound.

Example 9

The library production method of any one of (Example 1) to (Example 8)described above, wherein the aforementioned target tissue is a cancertissue or an inflammatory tissue.

Example 10

The library production method of (Example 9) described above, whereinthe cancer tissue-specific compound is at least one compound selectedfrom the group consisting of nucleosides that have a purine ringstructure, amino acids and their metabolites, lipids and theirmetabolites, primary metabolites from sugar metabolism, and nicotinamideand its metabolites.

Example 11

The library production method of any one of (Example 1) to (Example 10)described above, wherein the small molecule compound is kynurenine,adenosine, adenosine monophosphate, adenosine diphosphate, or adenosinetriphosphate.

Example 12

The library production method of any one of (Example 1) to (Example 11)described above, wherein the amino acid sites not involved in bindingwith the small molecule compound are sites other than any one or more ofthe amino acids selected from below:

H chain: 97, 100c, 101, 94, 95, 100d, 100e, 33, 50, 52, 56, 57, 58, 99,100, 100a, 54, 55 (Kabat Numbering); and

L chain: 49, 55, 95c, 96, 95a, 95b (Kabat Numbering).

Library Other Embodiments

An embodiment of a library of the present invention that can yieldantigen-binding domains whose antigen-binding ability varies dependingon the concentration of a small molecule compound is, for example, thelibrary below.

A library that comprises mainly:

(i) a plurality of antigen-binding domains or antigen-binding moleculescomprising an antigen-binding domain, which have different sequencesfrom one another; or

(ii) nucleic acids that encode a plurality of antigen-binding domains orantigen-binding molecules comprising an antigen-binding domain, whichhave different sequences from one another;

wherein the antigen-binding molecule domains or antigen-bindingmolecules are antigen-binding domains or antigen-binding moleculescomprising an antigen-binding domain having binding activity to a smallmolecule compound. A library of this embodiment preferably has diversityof 1.2×10⁸ or higher.

The term “comprises mainly” in the description of a library thatcomprises mainly a plurality of antigen-binding molecules in thisembodiment reflects the number of antigen-binding molecules havingbinding activity to a small molecule compound (for example, a targettissue-specific compound) among the number of independent clones thatdiffer in sequence in the library. Specifically, presence of at least10⁴ antigen-binding molecules that exhibit such binding activity in thelibrary is preferred. In other words, the term may be suitably expressedas the ratio of antigen-binding molecules in which the antigen-bindingactivity of the antigen-binding domain differs depending on the presenceor absence of the small molecule to the number of the independent clonesthat differ in sequence in the library. Specifically, theantigen-binding domains of the present invention can be obtained from alibrary that comprises antigen-binding molecules that exhibit suchbinding activity at a ratio of 10⁻⁶% to 80%, or 10⁻⁵% to 60%, preferably10⁻⁴% to 40%, more preferably 10⁻³% to 40%, and even more preferably10⁻²% to 40% to the number of the independent clones that differ insequence in the library. Similar to the case above, fusion polypeptides,polynucleotide molecules, or vectors can also be presented as the numberof molecules or the ratio to all molecules. In addition, viruses canalso be presented as the number of individual viruses or the ratio toall viruses as in the above case.

An embodiment of a library of the present invention that can yieldantigen-binding domains whose antigen-binding activity varies dependingon the concentration of a small molecule compound is, for example, thelibrary below.

A library that comprises mainly:

(i) a plurality of antibody molecules having different sequences fromone another; or

(ii) nucleic acids that encode a plurality of antibody molecules havingdifferent sequences from one another;

wherein the antibody molecules have binding activity to a small moleculecompound and have a diversity that fulfills any one of (i) to (vi)below:(i) heavy chain CDR1 diversity of 13 or higher;(ii) heavy chain CDR2 diversity of 129 or higher;(iii) heavy chain CDR3 diversity of 5 or higher;(iv) light chain CDR1 diversity of 193 or higher;(v) light chain CDR2 diversity of 7 or higher; and(vi) light chain CDR3 diversity of 17 or higher.

The term “comprises mainly” in the description of a library thatcomprises mainly a plurality of antigen-binding molecules in thisembodiment reflects the number of antigen-binding molecules havingbinding activity to a small molecule compound (for example, a targettissue-specific compound) among the number of independent clones thatdiffer in sequence in the library. Specifically, presence of at least10⁴ antigen-binding molecules that exhibit such binding activity in thelibrary is preferred. In other words, the term may be suitably expressedas the ratio of antigen-binding molecules in which the antigen-bindingactivity of the antigen-binding domain differs depending on the presenceor absence of the small molecule to the number of independent clonesthat differ in sequence in the library. Specifically, theantigen-binding domains of the present invention can be obtained from alibrary comprising antigen-binding molecules that exhibit such bindingactivity at a ratio of 10⁻⁶% to 80%, or 10⁻⁵% to 60%, preferably 10⁻⁴%to 40%, more preferably 10⁻³% to 40%, and even more preferably 10′% to40% to the number of independent clones that differ in sequence in thelibrary. As in the case above, fusion polypeptides, polynucleotidemolecules, or vectors can also be presented as the number of moleculesor the ratio to all molecules. In addition, similar to the case above,viruses can also be presented as the number of individual virusindividuals or the ratio to all viruses.

An embodiment of a library of the present invention that can yieldantigen-binding domains whose antigen-binding ability varies dependingon the concentration of a small molecule compound is, for example, thelibrary below.

A library that comprises mainly:

(i) a plurality of antigen-binding domains or antigen-binding moleculescomprising an antigen-binding domain, which have different sequencesfrom one another; or

(ii) nucleic acids that encode a plurality of antigen-binding domains orantigen-binding molecules comprising an antigen-binding domain, whichhave different sequences from one another;

wherein the antigen-binding molecule domains or antigen-bindingmolecules are antigen-binding domains or antigen-binding moleculescomprising an antigen-binding domain that have binding activity to asmall molecule compound, and the library is for obtainingantigen-binding domains or antigen-binding molecules comprising anantigen-binding domain whose antigen-binding activity varies dependingon the concentration of a small molecule compound.

The term “comprises mainly” in the description of a library thatcomprises mainly a plurality of antigen-binding molecules in thisembodiment reflects the number of antigen-binding molecules havingbinding activity to a small molecule compound (for example, a targettissue-specific compound) among the number of independent clones thatdiffer in sequence in the library. Specifically, presence of at least10⁴ antigen-binding molecules that exhibit such binding activity in thelibrary is preferred. In other words, the term may be suitably expressedas the ratio of antigen-binding molecules in which the antigen-bindingactivity of the antigen-binding domain differs depending on the presenceor absence of the small molecule to the number of independent clonesthat differ in sequence in the library. Specifically, theantigen-binding domains of the present invention can be obtained from alibrary comprising antigen-binding molecules that exhibit such bindingactivity at a ratio of 10⁻⁶% to 80%, or 10⁻⁵% to 60%, preferably 10⁻⁴%to 40%, more preferably 10⁻³% to 40%, and even more preferably 10⁻²% to40% to the number of independent clones that differ in sequence in thelibrary. As in the above case, fusion polypeptides, polynucleotidemolecules, or vectors can also be presented as the number of moleculesor the ratio to all molecules. In addition, viruses can also bepresented as the number of individual viruses or the ratio to allviruses as in the above case.

Amino Acids that Change the Antigen-Binding Activity of anAntigen-Binding Domain Depending on the Presence or Absence of a SmallMolecule

With respect to methods for obtaining (methods of screening for)templates (antigen-binding domains whose antigen-binding activity variesdepending on the concentration of a small molecule compound, orantigen-binding domains having binding activity to a small moleculecompound) to be used when producing a library of the present invention,these antigen-binding domains and such may be prepared in any manner. Itis possible to use pre-existing antigen-binding domains or antibodies,and pre-existing libraries (phage libraries, etc.), antibodies orlibraries prepared from hybridomas obtained by immunizing animals orfrom B cells of immunized animals, for example, antibodies or librariesprepared from immune cells such as B cells of animals immunized with aconjugate in which adenosine or ATP, which is an embodiment of the smallmolecule compound, is suitably linked to an adjuvant agent such as ahighly immunogenic T cell epitope peptide, without being limitedthereto. A non-limiting example of the T cell epitope peptide suitablyincludes a Tetanus toxin-derived p30 helper peptide (shown in SEQ ID NO:4, and also referred to as Fragment C (FrC)).

In a non-limiting embodiment of a method for preparing antigen-bindingdomains or antibodies of the present invention obtained by theaforementioned screening method, it is possible to use a library thatcomprises, for example, a module of about 35 amino acids called the Adomain which is contained in the in vivo cell membrane protein Avimer;Adnectin containing the 10Fn3 domain which binds to the protein infibronectin, a glycoprotein expressed on cell membranes; Affibody whichhas as scaffold the IgG-binding domain composed of a 58-amino acidthree-helix bundle of Protein A; Designed Ankyrin Repeat proteins(DARPins) which are regions exposed on the molecular surface of ankyrinrepeats (AR) having a structure of repeatedly stacked subunitsconsisting of a turn containing 33 amino acid residues, two antiparallelhelices, and a loop; Anticalins and such, in which four loop regionssupport one side of a barrel structure formed by eight antiparallelstrands twisted toward the center, which are highly conserved inlipocalin molecules such as neutrophil gelatinase-associated lipocalin(NGAL); and the concave region formed by the parallel-sheet structureinside the horseshoe-shaped structure constituted by stacked repeats ofthe leucine-rich-repeat (LRR) module of the variable lymphocyte receptor(VLR) which does not have the immunoglobulin structure and is used inthe acquired immune system in jawless vertebrates such as lamprey andhagfish.

Preferred antigen-binding domains of the present invention include, forexample, antigen-binding domains containing antibody heavy-chain andlight-chain variable regions. Preferred examples of such antigen-bindingdomains include “single chain Fv (scFv)”, “single chain antibody”, “Fv”,“single chain Fv 2 (scFv2)”, “Fab”, “F(ab)2”, and IgG, and a librarycomprising thereof may also be used.

Furthermore, as a non-limiting embodiment of a method for preparingantigen-binding domains or antibodies of the present invention obtainedby the aforementioned screening method, it is possible to use atechnique for preparing antigen-binding domains or antibodies havingbinding activity to a small molecule compound by panning using anabove-mentioned library. As a library, it is possible to use, forexample, but without being limited thereto, a phage display library, aribosome display library, an mRNA display library, a cDNA displaylibrary, a CIS display library, an E. coli display library, aGram-positive bacterium display library, an yeast display library, amammalian cell display library, a virus display library, and an in vitrovirus display library.

In an embodiment of the aforementioned technique for preparingantigen-binding domains or antibodies having binding activity to a smallmolecule compound by panning, small molecule compounds fixed onto acarrier such as beads can be used. The fixed small molecule compoundscan be produced by, for example, without being limited thereto, a methodof contacting small molecule compounds synthesized to be chemicallylinked to biotin via a linker with beads or a plate onto whichstreptavidin or NeutrAvidin has been fixed, or a method of adhering thesmall molecule compounds covalently linked to an adjuvant such as bovineserum albumin (BSA) to beads or plates by hydrophobic interaction. Thesemethods are already publicly known (J. Immunol. Methods. 2003 September,280 (1-2): 139-55; BMC Biotechnol. 2009 Jan. 29; 9: 6. doi:10.1186/1472-6750-9-6). Antigen-binding domains or antibodies havingbinding activity to the small molecule compounds can be prepared bycollecting antigen-binding domains or antibodies that have bindingactivity to the fixed small molecule compounds.

Alternatively, in another embodiment of the aforementioned technique forpreparing antigen-binding domains or antibodies having binding activityto a small molecule compound by panning, a fluorescence-labeled smallmolecule compound, or a biotin-labeled small molecule compound andfluorescence-labeled streptavidin (or NeutrAvidin or avidin) may beused. Antigen-binding domains or antibodies having binding activity tothe small molecule compound can be prepared by contacting thefluorescence-labeled small molecule compound, or the biotin-labeledsmall molecule compound and fluorescence-labeled streptavidin (orNeutrAvidin or avidin), with a library presented on the cell surface orsuch, and then using the fluorescence-activated cell sorting (FACS)method. These methods are already publicly known (Proc Natl Acad SciUSA. 2000 Sep. 26; 97 (20): 10701-5).

Furthermore, in a non-limiting embodiment of a method for preparingantigen-binding domains or antibodies of the present invention obtainedby the aforementioned screening method, pre-existing antigen-bindingdomains having binding activity to the small molecule compound may beused. For example, when adenosine and/ATP are used as an example,without being limited thereto, molecules belonging to the kinase familyhaving ATP-binding activity can be used as the antigen-binding domain,and molecules belonging to the adenosine deaminase family havingadenosine-binding activity can be used as the antigen-binding domain. Byproducing a library of portions in these molecules that are not involvedin the binding to ATP and/or adenosine, it is possible to obtainantigen-binding molecules that show antigen binding in a mannerdependent on the ATP and/or adenosine concentration.

As a method for obtaining antigen-binding domains usingnon-antibody-like proteins, a library produced by using the loop-formingsites and surface residues of the α-helix of the non-antibody-likeprotein can be used, without being limited thereto. Methods forconstructing such libraries are already known (Nat Biotechnol. 2004 May;22 (5): 575-82; J Mol Biol. 1998 Dec. 11; 284 (4): 1141-51; and NatBiotechnol. 1997 August; 15 (8): 772-7). Furthermore, techniques forobtaining antigen-binding domains that have binding activity to a smallmolecule compound by panning using a library constructed as describedabove are publicly known. In one example, the constructed library isexpressed on the surface of phages by the phage display method. Phagesexpressing binding domains that bind to a small molecule compound linkedto bovine serum albumin, biotin, or such may be selected using beads,immunotubes, plates, and such. Such methods for obtainingnon-antibody-like antigen-binding domains that have binding activity toa small molecule compound are already known (Proc Natl Acad Sci USA.1999 Mar 2; 96 (5): 1898-903). Furthermore, amino acid sites notinvolved in the binding to the small molecule compound and amino acidsites not important for canonical structure formation can be identifiedby, without being limited thereto, performing a crystal structureanalysis on these antigen-binding domains which have binding activity tothe small molecule compound, or by producing variants and evaluatingtheir binding activities (J Mol Biol. 2003 Jul. 4; 330 (2): 385-96,Proteins. 2003 Oct. 1; 53 (1): 121-9). The libraries described in thepresent invention can be constructed by introducing diversity to theamino acid sites identified in this manner. Furthermore, in anotherembodiment of the present invention, a library of limited amino acidsites can also be used. Anticalin has been reported as anon-antibody-like antigen-binding domain, and is a four-loop region thatsupports one side of a barrel structure formed by eight antiparallelstrands twisted toward the center, which are highly conserved inlipocalin molecules such as neutrophil gelatinase-associated lipocalin(NGAL). In Anticalin, the amino acid sites used for binding to a smallmolecule compound are known to be different from the amino acid sitesused for protein binding; and as an example, without being limitedthereto, it is known that mutually different libraries, in which aminoacid sites that can be involved in binding to the small molecule andamino acid sites that can be involved in binding to the protein aremutated respectively, can be used (FEBS Lett. 2014 Jan. 21;588(2):213-8). Therefore, it is possible to construct a library of thepresent invention by obtaining antigen-binding domains having bindingactivity to a small molecule compound from a library that can yieldbinding domains for the small molecule compound, and then introducingdiversity at the amino acid sites used to obtain antigen-binding domainshaving protein-binding activity to the obtained antigen-binding domainshaving binding activity to the small molecule compound. Morespecifically, it is known that in human lipocalin2 (Lcn2), each of theamino acid sites, V33, L36, I41, Y52, T54, S68, L70, W79, R81, K134,T136, and Y138, can be used as a site for introducing diversity in alibrary for obtaining small molecule compound-binding domains (J Am ChemSoc. 2009 Mar 18; 131(10):3565-76); and similarly, each of the aminoacid sites, A40, L42, E44, K46, D47, Q49, K50, L70, R72, K73, D77, W79,P101, G102, L103, K125, S127, Q128, R130, and Y132, can be used as asite for introducing diversity in a library for obtainingprotein-binding domains (Proc Natl Acad Sci USA. 2009 May 19;106(20):8198-203). Therefore, without being limited thereto, it ispossible to construct a library of the present invention by first usinga library that comprises antigen-binding domains made to have diversityat each of the amino acid sites, V33, L36, I41, Y52, T54, S68, L70, W79,R81, K134, T136, and Y138, to obtain antigen-binding domains havingbinding activity to a small molecule compound, and then introducingdiversity to the obtained antigen-binding domains having bindingactivity to the small molecule compound at each of the amino acid sites,A40, L42, E44, K46, D47, Q49, K50, R72, K73, D77, P101, G102, L103,K125, S127, Q128, R130, and Y132. For antibodies and non-antibody-likeantigen-binding domains other than lipocalin molecules, those skilled inthe art can also construct libraries of the present invention byappropriately referring to the above-described library constructionmethods. In another embodiment, antigen-binding domains having bindingactivity to a small molecule compound can be used. As an example,Rhodnius prolixus aggregation inhibitor 1 (RPAI-1) belonging to thelipocalin family, which is known to have binding activity to ATP, ADP,AMP, and adenosine, can be used (J Biol Chem. 2000 Apr. 28; 275 (17):12639-50 and Biochemistry, 2002 Mar. 19; 41 (11): 3810-8). Amino acidsites not involved in the binding to the small molecule compound andamino acid sites not important for canonical structure formation can beidentified by analyzing the crystal structure of the antigen-bindingdomains, or by producing variants and then evaluating their bindingactivities, without being limited thereto. Libraries of the presentinvention can be constructed by introducing diversity at the amino acidsites identified in this manner. Presence of antigen-binding domainsbelonging to the lipocalin family having binding activity to varioussmall molecule compounds besides ATP, ADP, AMP, and adenosine, such ashistamine, serotonin, adrenaline, and noradrenalin, are known (J BiolChem. 2003 Feb. 14; 278 (7): 4611-7 and Expert Rev Clin Immunol. 2007July; 3 (4): 491-501); and without being limited thereto, they can beused to construct libraries of the present invention that useantigen-binding domains having binding activity to various smallmolecule compounds. For other non-antibody-like antigen-binding domainsand antibodies, libraries of the present invention can also be producedby those skilled in the art by appropriately referring to theabove-described library construction methods.

As a non-limiting embodiment of the present invention, detaileddescription will be made using adenosine and/or ATP as examples, but thefollowing examples are also appropriately applied to small moleculesbesides adenosine and/or ATP. Examples of amino acids that change theantigen-binding activity of the antigen-binding molecule depending onthe presence or absence of adenosine and/or ATP as described above mayinclude amino acids that form an adenosine- and/or ATP-binding motif.The amino acid positions where the above-mentioned amino acids arecontained in the antigen-binding domain are not limited to any specificposition, and as long as the antigen-binding activity of theantigen-binding domain changes depending on the presence or absence ofadenosine and/or ATP, any position in the heavy chain variable region orlight chain variable region forming the antigen-binding domain ispossible. More specifically, the antigen-binding domains of the presentinvention may be obtained from a library comprising mainlyantigen-binding molecules that have different sequences from oneanother, in which amino acids that change the antigen-binding activityof the antigen-binding molecule depending on the presence or absence ofadenosine and/or ATP are contained in the antigen-binding domain of theheavy chain. In a non-limiting embodiment, antigen-binding domains ofthe present invention may be obtained from a library comprising mainlyantigen-binding molecules that have different sequences from oneanother, in which amino acids that change the antigen-binding activityof the antigen-binding molecule depending on the presence or absence ofadenosine and/or ATP are contained in CDR1, CDR2, and/or CDR3 of theheavy chain. In another non-limiting embodiment, antigen-binding domainsof the present invention may be obtained from a library mainlycomprising antigen-binding molecules having different sequences from oneanother, in which the amino acids that change the antigen-bindingactivity of the antigen-binding molecule depending on the presence orabsence of adenosine and/or ATP are contained in FR1, FR2, FR3 and/orFR4 of the heavy chain.

Furthermore, in an embodiment of the present invention, antigen-bindingdomains of the present invention may be obtained from a library mainlycomprising antigen-binding molecules having different sequences from oneanother, in which the amino acids that change the antigen-bindingactivity of the antigen-binding molecule depending on the presence orabsence of adenosine and/or ATP are contained in the antigen-bindingdomain of the heavy chain and/or light chain. In a non-limitingembodiment, antigen-binding domains of the present invention may beobtained from a library mainly comprising antigen-binding moleculeshaving different sequences from one another, in which the amino acidsthat change the antigen-binding activity of the antigen-binding moleculedepending on the presence or absence of adenosine and/or ATP arecontained in CDR1, CDR2, and/or CDR3 of the heavy chain and/or lightchain. In another non-limiting embodiment, antigen-binding domains ofthe present invention may be obtained from a library mainly comprisingantigen-binding molecules having different sequences from one another,in which the amino acids that change the antigen-binding activity of theantigen-binding molecule depending on the presence or absence ofadenosine and/or ATP are contained in FR1, FR2, FR3 and/or FR4 of theheavy chain and/or light chain.

In a non-limiting embodiment, examples of such amino acids include anyone or more amino acids selected from amino acids at positions 52, 52a,53, 96, 100a, and 100c contained in the heavy chain variable region.Also, in a non-limiting embodiment, examples of such amino acids includeone or more amino acids selected from amino acids including Ser atposition 52, Ser at position 52a, Arg at position 53, Gly at position96, Leu at position 100a, and Trp at position 100c contained in theheavy chain variable region.

Any framework sequence can be used as the framework sequence of thelight-chain and/or heavy-chain variable regions of an antigen-bindingmolecule as long as the amino acids that change the antigen-bindingactivity of the antigen-binding molecule depending on the presence orabsence of adenosine and/or ATP are contained in the antigen-bindingdomain of the heavy chain and/or light chain. The origin of theframework sequences is not limited, and they may be obtained from humanor any nonhuman organisms. Such organisms preferably include mice, rats,guinea pigs, hamsters, gerbils, cats, rabbits, dogs, goats, sheep,bovines, horses, camels and organisms selected from nonhuman primates.In a particularly preferred embodiment, the framework sequences of thelight chain and/or heavy chain variable region of an antigen-bindingmolecule preferably have human germ-line framework sequences. Thus, inan embodiment of the present invention, if the entire frameworksequences are human sequences, it is thought that an antigen-bindingmolecule of the present invention induces little or no immunogenicresponse when it is administered to humans (for example, to treatdiseases). In the above sense, the phrase “containing a germ linesequence” in the present invention means that a part of the frameworksequences of the present invention is identical to a part of any humangerm line framework sequences. Specifically, the framework sequence ofthe present invention is at least 50% or more, 60% or more, 70% or more,80% or more, 90% or more, or 100% or more identical to the germ linesequence. For example, when the heavy chain FR2 sequence of anantigen-binding molecule of the present invention is a combination ofheavy chain FR2 sequences of different human germ line frameworksequences, such a molecule is also an antigen-binding molecule“containing a germ line sequence” in the present invention. Even whenthe framework sequences of antigen-binding molecules of the presentinvention are sequences with substitutions, they are antigen-bindingmolecules “containing a germ line sequence” of the present invention.Examples of such sequences with substitutions include, in particular,sequences in which amino acids of part of human germ line frameworksequences have been substituted with amino acids that change theantigen-binding activity of the antigen-binding molecule depending onthe presence or absence of adenosine and/or ATP.

Preferred examples of the frameworks include, for example, fully humanframework region sequences currently known, which are included in thewebsite of V-Base (http://vbase.mrc-cpe.cam.ac.uk/) or others. Thoseframework region sequences can be appropriately used as a germ linesequence contained in an antigen-binding molecule of the presentinvention. The germ line sequences may be categorized according to theirsimilarity (Tomlinson et al. (J. Mol. Biol. (1992) 227, 776-798);Williams and Winter (Eur. J. Immunol. (1993) 23, 1456-1461); Cox et al.(Nat. Genetics (1994) 7, 162-168)). Appropriate germ line sequences canbe selected from Vκ, which is grouped into seven subgroups; Vλ, which isgrouped into ten subgroups; and VH, which is grouped into sevensubgroups.

Fully human VH sequences preferably include, but are not limited to, forexample, VH sequences of:

subgroup VH1 (for example, VH1-2, VH1-3, VH1-8, VH1-18, VH1-24, VH1-45,VH1-46, VH1-58, and VH1-69);subgroup VH2 (for example, VH2-5, VH2-26, and VH2-70);subgroup VH3 (VH3-7, VH3-9, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20,VH3-21, VH3-23, VH3-30, VH3-33, VH3-35, VH3-38, VH3-43, VH3-48, VH3-49,VH3-53, VH3-64, VH3-66, VH3-72, VH3-73, and VH3-74);subgroup VH4 (VH4-4, VH4-28, VH4-31, VH4-34, VH4-39, VH4-59, andVH4-61);subgroup VH5 (VH5-51);subgroup VH6 (VH6-1); andsubgroup VH7 (VH7-4 and VH7-81).These are also described in known documents (Matsuda et al. (J. Exp.Med. (1998) 188, 1973-1975)) and such, and thus persons skilled in theart can appropriately design antigen-binding molecules of the presentinvention based on the information of these sequences. It is alsopreferable to use other fully human frameworks or framework sub-regions.

Fully human Vκ sequences preferably include, but are not limited to, forexample:

A20, A30, L1, L4, L5, L8, L9, L11, L12, L14, L15, L18, L19, L22, L23,L24, O2, O4, O8, O12, O14, and O18 grouped into subgroup Vk1;A1, A2, A3, A5, A7, A17, A18, A19, A23, O1, and O11, grouped intosubgroup Vk2;A11, A27, L2, L6, L10, L16, L20, and L25, grouped into subgroup Vk3;B3, grouped into subgroup Vk4;B2 (herein also referred to as Vk5-2), grouped into subgroup Vk5; andA10, A14, and A26, grouped into subgroup Vk6(Kawasaki et al. (Eur. J. Immunol. (2001) 31, 1017-1028); Schable andZachau (Biol. Chem. Hoppe Seyler (1993) 374, 1001-1022);Brensing-Kuppers et al. (Gene (1997) 191, 173-181)).

Fully human Vλ sequences preferably include, but are not limited to, forexample:

V1-2, V1-3, V1-4, V1-5, V1-7, V1-9, V1-11, V1-13, V1-16, V1-17, V1-18,V1-19, V1-20, and V1-22, grouped into subgroup VL1;V2-1, V2-6, V2-7, V2-8, V2-11, V2-13, V2-14, V2-15, V2-17, and V2-19,grouped into subgroup VL1;V3-2, V3-3, and V3-4, grouped into subgroup VL3;V4-1, V4-2, V4-3, V4-4, and V4-6, grouped into subgroup VL4; andV5-1, V5-2, V5-4, and V5-6, grouped into subgroup VL5 (Kawasaki et al.(Genome Res. (1997) 7, 250-261)).

Normally, these framework sequences are different from one another atone or more amino acid residues. These framework sequences can be usedin combination with “at least one amino acid residue that alters theantigen-binding activity of an antigen-binding domain depending on thepresence or absence of adenosine and/or ATP” of the present invention.Other examples of the fully human frameworks used in combination with“at least one amino acid residue that alters the antigen-bindingactivity of an antigen-binding domain depending on the presence orabsence of adenosine and/or ATP” of the present invention include, butare not limited to, for example, KOL, NEWM, REI, EU, TUR, TEI, LAY, andPOM (for example, Kabat et al. (1991) supra; Wu et al. (J. Exp. Med.(1970) 132, 211-250)).

Without being bound by a particular theory, one reason for theexpectation that the use of germ line sequences precludes adverse immuneresponses in most individuals is believed to be as follows. As a resultof the process of affinity maturation during normal immune responses,somatic mutation occurs frequently in the variable regions ofimmunoglobulin. Such mutations mostly occur around CDRs whose sequencesare hypervariable, but also affect residues of framework regions. Suchframework mutations do not exist on the germ line genes, and also theyare less likely to be immunogenic in patients. On the other hand, thenormal human population is exposed to most of the framework sequencesexpressed from the germ line genes. As a result of immunotolerance,these germ line frameworks are expected to have low or no immunogenicityin patients. To maximize the possibility of immunotolerance, variableregion-encoding genes may be selected from a group of commonly occurringfunctional germ line genes.

Known methods such as site-directed mutagenesis (Kunkel et al. (Proc.Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR canbe appropriately employed to produce the antigen-binding molecules ofthe present invention in which the above-described variable regionsequences, heavy or light chain variable region sequences, CDRsequences, or framework sequences contain amino acids that alter theantigen-binding activity of the antigen-binding domain depending on thepresence or absence of adenosine and/or ATP.

For example, a library which contains a plurality of antigen-bindingmolecules of the present invention whose sequences are different fromone another can be constructed by combining heavy chain variable regionsprepared as a randomized variable region sequence library with a lightchain variable region selected as a CDR sequence and/or frameworksequence originally containing at least one amino acid residue thatalters the antigen-binding activity of the antigen-binding domaindepending on the presence or absence of adenosine and/or ATP.

Alternatively, a heavy chain and/or light chain variable region sequenceselected as a CDR sequence and/or a framework sequence originallycontaining at least one amino acid residue that changes theantigen-binding activity of an antigen-binding domain depending on thepresence or absence of adenosine and/or ATP as mentioned above, can bedesigned to contain various amino acid residues other than the aboveamino acid residue(s). Herein, such residues are referred to as“flexible residues”. The number and position of flexible residues arenot particularly limited as long as the antigen-binding activity of theantigen-binding molecule of the present invention varies depending onthe concentration of a tissue-specific compound. Specifically, the CDRsequences and/or FR sequences of the heavy chain and/or light chain maycontain one or more flexible residues. One can identify the flexibleresidues and the amino acids that those residues can be substituted withfor library production by introducing mutations or by crystal structureanalysis of complexes formed by an antibody and adenosine and/or ATP.For example, from crystal structure analysis of complexes formed by anantibody and adenosine and/or ATP, one can identify residues in theantibody that are not involved in binding to adenosine and/or ATP. Onecan select amino acids that can maintain binding to the compounds at anappropriate level even when the residues that have been identified asnot being involved in binding to adenosine and/or ATP are substitutedwith those amino acids. Accordingly, it is possible to design a librarythat has the selected amino acids for the identified residues. In thiscase, one can design a library mainly comprising multipleantigen-binding molecules to be an assembly of antigen-binding moleculesin which residues identified as not being involved in binding toadenosine and/or ATP have been substituted with amino acids that aredifferent from one another. That is, the combination of individualflexible residues substituted with amino acids that are different fromone another can provide sequence diversity in antigen-binding moleculescontaining the flexible residues.

Antigen-binding molecules can be designed to include residues wherein atleast one of the residues that are identified to be involved in bindingto adenosine and/or ATP binding becomes any residue selected from thatresidue and other residues that are different from that residue. In anon-limiting embodiment, examples of amino acids that are identified asbeing involved in binding to adenosine and/or ATP may include one ormore amino acids selected from amino acids at positions 52, 52a, 53, 96,100a, and 100c in the heavy chain variable region. In a non-limitingembodiment, examples of such amino acids include one or more amino acidsselected from amino acids including Ser at position 52, Ser at position52a, Arg at position 53, Gly at position 96, Leu at position 100a, andTrp at position 100c contained in the heavy chain variable region. Forexample, when Leu at position 100a mentioned above is identified to beinvolved in binding to adenosine and/or ATP, the amino acid residue atposition 100a in the antigen-binding molecules included in the librarymay be any amino acid residue selected from the flexible residues ofHis, Met, Leu, Arg, Trp, or Tyr, in addition to Leu.

In a non-limiting embodiment, examples of the flexible residues mayinclude amino acids at positions 31, 32, 33, 35, 50, 55, 56, 57, 58, 59,95, 96, 97, 98, 99, 100, 100a, and 100b contained in the heavy chainvariable region. In another non-limiting embodiment, examples of suchamino acids may include amino acids at positions 26, 27, 27a, 27b, 27c,28, 29, 31, 32, 50, 51, 52, 53, 54, 55, 89, 90, 91, 92, 93, 94, 95a, 96,and 97 contained in the light chain variable region.

In a non-limiting embodiment, examples of the aforementioned flexibleresidues may include the following amino acids contained in the heavychain variable region:

Asp, Gly, Asn, Ser, Arg, or Thr for the amino acid at position 31;Ala, Phe, His, Asn, Ser, or Tyr for the amino acid at position 32;Ala, Glu, Asp, Gly, Phe, Ile, His, Lys, Met, Leu, Asn, Gln, Pro, Ser,Arg, Trp, Val, Tyr, or Thr for the amino acid at position 33;His, Ser, Thr, Tyr, or Asn for the amino acid at position 35;Ala, Glu, Asp, Gly, Phe, Ile, His, Lys, Met, Leu, Asn, Gln, Pro, Arg,Thr, Trp, Val, Tyr, or Ser for the amino acid at position 50;Ala, Glu, Asp, Gly, Leu, Thr, Ser, Arg, or Asn for the amino acid atposition 55;Ala, Glu, Asp, Gly, Phe, Ile, His, Lys, Met, Leu, Gln, Pro, Ser, Thr,Trp, Val, or Tyr for the amino acid at position 56;Ala, Lys, Arg, Thr, or Ile for the amino acid at position 57;Asp, Gly, Phe, His, Ser, Thr, Tyr, or Asn for the amino acid at position58;Leu, or Tyr for the amino acid at position 59;Ala, Ile, Lys, Met, Leu, Arg, Trp, Val, Tyr, or Phe for the amino acidat position 95;Ala, Asp, Asn, or Ser for the amino acid at position 96;Ala, Asp, Gly, Ile, His, Lys, Met, Leu, Asn, Ser, Val, Tyr, or Arg forthe amino acid at position 97;Ala, Glu, Asp, Gly, Phe, Ile, His, Met, Leu, Asn, Gln, Pro, Ser, Arg,Thr, Trp, Val, Tyr, or Lys for the amino acid at position 98;Ala, Glu, Asp, Phe, His, Lys, Asn, Gln, Ser, Arg, Trp, Val, Tyr, or Glyfor the amino acid at position 99;Ala, Glu, Gly, Phe, Ile, His, Lys, Met, Leu, Asn, Gln, Pro, Ser, Arg,Thr, Trp, Val, Tyr, or Asp for the amino acid at position 100;Ala, Phe, Ile, His, Lys, Met, Arg, Trp, Val, or Tyr for the amino acidat position 100a; orAla, Glu, Asp, Gly, Phe, Ile, His, Lys, Met, Leu, Gln, Pro, Ser, Arg,Thr, Trp, Val, Tyr, or Asn for the amino acid at position 100b.

In a non-limiting embodiment, examples of the aforementioned flexibleresidues may include the following amino acids contained in the lightchain variable region:

Ala, Ser, or Thr for the amino acid at position 26;Thr or Ser for the amino acid at position 27;Gly, Asn, Thr, or Ser for the amino acid at position 27a;Asn or Asp for the amino acid at position 27b;Ile or Val for the amino acid at position 27c;Asp or Gly for the amino acid at position 28;Ala, Asp, Phe, Ser, Arg, Thr, Tyr, or Gly for the amino acid at position29;Glu, Asp, Lys, or Asn for the amino acid at position 31;Ala, Asp, Ser, Thr, or Tyr for the amino acid at position 32;Asp, Gly, Lys, Asn, Gln, Ser, Arg, Tyr, or Glu for the amino acid atposition 50;Asp, Gly, Lys, Asn, Thr, or Val for the amino acid at position 51;Ala, Asp, Asn, Thr, or Ser for the amino acid at position 52;Glu, Asp, His, Asn, Gln, Ser, Tyr, or Lys for the amino acid at position53;Lys or Arg for the amino acid at position 54;Leu or Pro for the amino acid at position 55;Ala, Gly, Phe, Leu, Asn, Gln, Thr, Val, Tyr, or Ser for the amino acidat position 89;Ala, Leu, Thr, Val, or Ser for the amino acid at position 90;Ala, Asp, Phe, His, Lys, Asn, Ser, Arg, Thr, Trp, Val, or Tyr for theamino acid at position 91;Glu, Asp, Ser, Arg, Thr, Val, Tyr, or Ala for the amino acid at position92;Ala, Asp, Ile, Asn, Ser, Arg, Thr, Val, Tyr, or Gly for the amino acidat position 93;Ala, Asp, Gly, Ile, Asn, Arg, Thr, or Ser for the amino acid at position94;Ala, Glu, Asp, Gly, Phe, Ile, His, Lys, Met, Leu, Gln, Pro, Ser, Arg,Thr, Trp, Val, Tyr, or Asn for the amino acid at position 95;Ala, Glu, Asp, Gly, Ile, His, Lys, Leu, Gln, Pro, Ser, Arg, Thr, Tyr, orAsn for the amino acid at position 95a;Ala, Asp, Gly, Phe, His, Lys, Leu, Asn, Gln, Pro, Ser, Thr, Trp, Tyr, orVal for the amino acid at position 96; orAla, Gly, Ile, Met, Leu, Ser, or Val for the amino acid at position 97.

In an embodiment of the present invention, when the small moleculecompound is kynurenine, one can identify the flexible residues and theamino acids that those residues can be substituted with for libraryproduction by introduction of mutations and crystal structure analysisof complexes formed by an antibody and kynurenine. For example, fromcrystal structure analysis of complexes formed by an antibody andkynurenine, residues of the antibody that are not involved in kynureninebinding can be identified. One can select amino acids that can maintainan appropriate level of binding to the compound even when the residuesthat have been identified as not being involved in kynurenine bindingare substituted with those amino acids. Accordingly, it is possible todesign a library to have selected amino acids at the identifiedresidues. In this case, one can design a library to comprise mainly aplurality of antigen-binding molecules that is an assembly ofantigen-binding molecules in which residues identified as not beinginvolved in kynurenine binding have been substituted with amino acidsthat are different from one another. That is, combining each of theflexible residues that have been substituted with mutually differentamino acids can provide sequence diversity in antigen-binding moleculescontaining the flexible residues.

Antigen-binding molecules can be designed to include residues wherein atleast one of the residues that are identified to be involved inkynurenine binding becomes any residue selected from that residue andresidues different from that residue. In a non-limiting embodiment ofamino acids identified as being involved in kynurenine binding, examplesof amino acid residues whose side chains are involved in kynureninebinding may include any one or more amino acids selected from aminoacids at positions P97, R100c, and D101 (Kabat numbering) in the H chainand at positions H49 and F55 (Kabat numbering) in the L chain. Examplesof amino acid residues greatly involved in the binding with kynurenineat the main chain portion may include any one or more amino acidsselected from amino acids at positions R94, D95, R100c, G100d, and A100ein the H chain. Examples of residues important for maintaining thestructure of the H-chain CDR3 in a kynurenine-bound conformation asdetermined by X-ray crystallography may include any one or more aminoacids selected from amino acids at positions P97, P100b, and G100d inthe H chain.

In the present invention, a non-limiting embodiment of flexible residueswhen the small molecule is kynurenine may include, for example, aminoacids at positions 24, 26, 27, 28, 29, 30, 31, 32, 33, 50, 51, 52, 52a,53, 54, 55, 56, 58, 73, 95, 96, 97, 98, 99, 100, 100a, 100b, 100c, 100d,100e, 100f, and 102 contained in the heavy chain variable region. Inanother non-limiting embodiment, examples of such amino acids mayinclude amino acids at positions 27d, 27e, 28, 29, 32, 46, 49, 50, 51,52, 53, 54, 55, 92, 93, and 94 contained in the light chain variableregion.

A non-limiting embodiment of the aforementioned flexible residues is anamino acid contained in the heavy chain variable region, which is anyof:

Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,Ser, Thr, Val, Trp, or Tyr for the amino acid at position 24;Gly, Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Pro, Gln, Arg, Ser, Thr,Val, Trp, or Tyr for the amino acid at position 26;Gly, Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid at position 27;Thr, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Pro, Gln, Arg, Ser,Val, Trp, or Tyr for the amino acid at position 28;Phe, Ile, Leu, Trp, or Tyr for the amino acid at position 29;Ser, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln,Arg, Thr, Val, Trp, or Tyr for the amino acid at position 30;Ser, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Gln, Arg, Thr,Val, Trp, or Tyr for the amino acid at position 31;Tyr, Phe, or His for the amino acid at position 32;Ala, Gly, Ile, Lys, Gln, Arg, Ser, Thr, Val, or Trp for the amino acidat position 33;Gly, Ala, Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val,Trp, or Tyr for the amino acid at position 50;Ile, Ala, Gly, Lys, Leu, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for theamino acid at position 51;Ile, Ala, Glu, Phe, His, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Val,Trp, or Tyr for the amino acid at position 52;Pro, Ala, Gly, Ser, Thr, or Trp for the amino acid at position 52a;Ile, Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Asn, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid at position 53;Phe, Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Pro, Gln, Arg, Ser, Thr,Val, Trp, or Tyr for the amino acid at position 54;Gly, Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid at position 55;Thr, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg,Ser, Val, Trp, or Tyr for the amino acid at position 56;Asn, Ala, Asp, Glu, Phe, Gly, His, Lys, Pro, Gln, Arg, Ser, Thr, Val,Trp, or Tyr for the amino acid at position 58;Glu, Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser,Thr, Val, or Tyr for the amino acid at position 73;Asp or Gly for the amino acid at position 95;Ala, Glu, Phe, His, Ile, Lys, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyrfor the amino acid at position 96;Pro, Ala, Asn, or Ser for the amino acid at position 97;Val, Leu, or Thr for the amino acid at position 98;Val, Ala, Asp, Phe, His, Ile, Lys, Leu, Asn, Gln, Arg, Ser, Thr, or Tyrfor the amino acid at position 99;Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid at position 100;Arg, Ala, Asp, Glu, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, or Val forthe amino acid at position 100a;Pro, Ala, Lys, Asn, Gln, Arg, or Ser for the amino acid at position100b;Arg, His, Lys, or Gln for the amino acid at position 100c;Gly or Asn for the amino acid at position 100d;Ala, Gly, or Ser for the amino acid at position 100e;Phe or Leu for the amino acid at position 100f; orIle, Ala, Asp, Glu, Phe, His, Leu, Met, Asn, Gln, Arg, Thr, Val, Trp, orTyr for the amino acid at position 102.

A non-limiting embodiment of the aforementioned flexible residues is anamino acid contained in the light chain variable region, which is anyof:

His, Ala, Asp, Glu, Phe, Gly, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid at position 27d;Ser, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg,Thr, Val, Trp, or Tyr for the amino acid at position 27e;Asp, Ala, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid at position 28;Gly, Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Pro, Gln, Arg, Ser, Thr,Val, Trp, or Tyr for the amino acid at position 29;Tyr, Ala, Phe, Gly, His, Lys, Leu, Pro, Gln, Arg, Val, or Trp for theamino acid at position 32;Leu, Ile, Met, Asn, or Val for the amino acid at position 46;Tyr, Phe, His, or Trp for the amino acid at position 49;Glu, Ala, Phe, Gly, Ile, Lys, Leu, Met, Gln, Ser, Thr, Val, or Tyr forthe amino acid at position 50;Ile, Ala, Asp, Glu, Phe, Gly, Lys, Leu, Met, Gln, Arg, Ser, Thr, Val,Trp, or Tyr for the amino acid at position 51;Ser, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Gln, Arg, Thr,Val, Trp, or Tyr for the amino acid at position 52;Asn, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid at position 53;Arg, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser,Thr, Val, Trp, or Tyr for the amino acid at position 54;Phe, Leu, Met, Arg, or Tyr for the amino acid at position 55;Thr, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Gln, Arg, Ser,Val, Trp, or Tyr for the amino acid at position 92;Gln, Ala, Asp, Glu, Phe, Gly, Ile, Lys, Leu, Asn, Arg, Ser, Thr, Val, orTyr for the amino acid at position 93; orPhe, His, Ile, Lys, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for theamino acid at position 94.

In the present invention, another non-limiting embodiment of theflexible residues when the small molecule is kynurenine may include, forexample, amino acids at positions 28, 31, 33, 50, 51, 52, 54, 55, 56,58, 96, 97, 99, 100, 100a, 100b, and 100c contained in the heavy chainvariable region. In another non-limiting embodiment, examples of suchamino acids may include amino acids at positions 27d, 27e, 28, 29, 32,52, 53, 54, 56, 92, and 93 contained in the light chain variable region.

In an embodiment of the present invention, when the small moleculecompound is adenosine, one can identify the flexible residues and theamino acids that those residues can be substituted with for libraryproduction by introduction of mutations and crystal structure analysisof complexes formed by an antibody and adenosine. For example, residuesof the antibody that are not involved in adenosine binding can beidentified by crystal structure analysis of complexes formed by anantibody and adenosine. One can select amino acids that can maintain anappropriate level of binding to the compound even when the residues thathave been identified as not being involved in adenosine binding aresubstituted with those amino acids. Accordingly, it is possible todesign a library to have selected amino acids at the identifiedresidues. In this case, one can design a library to comprise mainlymultiple antigen-binding molecules to be an assembly of antigen-bindingmolecules in which residues identified as not being involved inadenosine binding have been substituted with amino acids that aredifferent from one another. That is, combining each of the flexibleresidues that have been substituted with mutually different amino acidscan provide sequence diversity in antigen-binding molecules containingthe flexible residues.

Antigen-binding molecules may be designed to include residues wherein atleast one of the residues that are identified to be involved inadenosine binding becomes any residue selected from that residue andresidues different from that residue. In a non-limiting embodiment ofamino acids that are identified as being involved in adenosine binding,examples may include any one or more amino acids selected from aminoacids at positions A33, I50, G52, S56, T57, W58, G99, Y100, and T100a(Kabat numbering) in the H chain and at positions Y95c and N96 (Kabatnumbering) in the L chain.

In the present invention, a non-limiting embodiment of flexible residueswhen the small molecule is adenosine may include, for example, aminoacids at positions 31, 32, 53, 54, 55, 56, 57, 59, 61, 62, 65, 96, 97,98, 100, 100a, 101, and 102 contained in the heavy chain variableregion. In another non-limiting embodiment, examples of such amino acidsmay include amino acids at positions 28, 29, 32, 93, 94, 95, 95a, 95b,and 95c contained in the light chain variable region.

A non-limiting embodiment of the aforementioned flexible residues is anamino acid contained in the heavy chain variable region, which is anyof:

Asn, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid at position 31;Tyr, Phe, Gly, His, Ile, Lys, Asn, Pro, Gln, Arg, Thr, Val, or Trp forthe amino acid at position 32;Ala, Asp, Glu, Phe, Gly, Ile, Lys, Leu, Pro, Gln, Arg, Ser, Thr, Val, orTyr for the amino acid at position 53;Asp, Glu, Phe, Gly, His, Ile, Leu, Gln, Ser, Thr, Val, or Tyr for theamino acid at position 54;Ser, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Gln, Arg, Thr, Val, orTyr for the amino acid at position 55;Ser, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Gln, Arg, Thr, orVal for the amino acid at position 56;Thr, Ala, Ile, Lys, Leu, Asn, Gln, Arg, Ser, or Val for the amino acidat position 57;Tyr, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Gln, Arg, Ser, Thr,Val, or Trp for the amino acid at position 59;Ser, Ala, Phe, His, Lys, Leu, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyrfor the amino acid at position 61;Trp, Ala, Asp, Glu, Phe, or Gly for the amino acid at position 62;Gly, Ala, Asp, Glu, Phe, Ile, Lys, Leu, Asn, Gln, Arg, Thr, Val, or Trpfor the amino acid at position 65;Arg, Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Asn, Gln, Ser, Thr, Val,Trp, or Tyr for the amino acid at position 96;Phe, Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Asn, Gln, Arg, Ser, Thr,Val, Trp, or Tyr for the amino acid at position 97;Val, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg,Ser, or Thr for the amino acid at position 98;Tyr or Phe for the amino acid at position 100;Thr, Ser, or Val for the amino acid at position 100a;Asp, Ala, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Gln, Arg, Ser, Thr,Val, Trp, or Tyr for the amino acid at position 101; orPro, Asp, or Asn for the amino acid at position 102.

A non-limiting embodiment of the aforementioned flexible residues is anamino acid contained in the light chain variable region, which is anyof:

Trp, Ala, Phe, His, Lys, Asn, Ser, Thr, Val, or Tyr for the amino acidat position 28;Asn, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid at position 29;Tyr, Ala, Asp, Phe, Gly, or His for the amino acid at position 32;Ala, Asp, Glu, Phe, Gly, His, Leu, Gln, Arg, Ser, Thr, Val, or Tyr forthe amino acid at position 93;Asn, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid at position 94;Ser, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg,Thr, Val, Trp, or Tyr for the amino acid at position 95;Gly, Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Val,Trp, or Tyr for the amino acid at position 95a;Trp, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg,Ser, Thr, Val, or Tyr for the amino acid at position 95b; orTyr, Phe, His, Lys, Leu, Asn, or Val for the amino acid at position 95c.

In an embodiment of the present invention, when the small moleculecompound is adenosine monophosphate, one can identify the flexibleresidues and the amino acids that those residues can be substituted withfor library production by crystal structure analysis of complexes formedby an antibody and adenosine monophosphate, and by introduction ofmutations and modeling based on the crystal structure of a complexformed by an antibody and adenosine which is an analogous compound. Forexample, residues of the antibody that are not involved in the bindingto adenosine monophosphate can be identified by modeling using thecrystal structure analysis of complexes formed by the antibody andadenosine. One can select amino acids that can maintain an appropriatelevel of binding to the compound even when the residues that have beenidentified as not being involved in adenosine monophosphate binding aresubstituted with those amino acids. Accordingly, it is possible todesign a library to have selected amino acids at the identifiedresidues. In this case, one can design a library to comprise mainly aplurality of antigen-binding molecules that is an assembly ofantigen-binding molecules in which residues identified as not beinginvolved in adenosine monophosphate binding have been substituted withamino acids that are different from one another. That is, combining eachof the flexible residues that have been substituted with mutuallydifferent amino acids can provide sequence diversity in antigen-bindingmolecules containing the flexible residues.

Antigen-binding molecules may be designed to include residues wherein atleast one of the residues that are identified to be involved inadenosine monophosphate binding becomes any residue selected from thatresidue and residues different from that residue. In a non-limitingembodiment of amino acids identified as being involved in the binding tothe ribose moiety and adenine ring moiety of adenosine monophosphate,examples may include any one or more amino acids selected from aminoacids at positions A33, I50, G52, S56, T57, W58, G99, Y100, and T100a(Kabat numbering) in the H chain and at positions Y95c and N96 (Kabatnumbering) in the L chain. In a non-limiting embodiment, examples ofamino acids identified as being involved in binding to the phosphatemoiety of adenosine monophosphate may include any one or more aminoacids selected from amino acids at positions D54, S55, S56, T57, and W58in the H chain CDR2 and at positions G95a, W95b, and Y95c (Kabatnumbering) in the L chain CDR3.

A non-limiting embodiment of the aforementioned flexible residues is anamino acid contained in the heavy chain variable region, which is anyof:

Asn, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid at position 31;Tyr, Phe, Gly, His, Ile, Lys, Asn, Pro, Gln, Arg, Thr, Val, or Trp forthe amino acid at position 32;Ala, Asp, Glu, Phe, Gly, Ile, Lys, Leu, Pro, Gln, Arg, Ser, Thr, Val, orTyr for the amino acid at position 53;Asp, Glu, Phe, Gly, His, Ile, Leu, Gln, Ser, Thr, Val, or Tyr for theamino acid at position 54;Ser, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Gln, Arg, Thr, Val, orTyr for the amino acid at position 55;Ser, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Gln, Arg, Thr, orVal for the amino acid at position 56;Thr, Ala, Ile, Lys, Leu, Asn, Gln, Arg, Ser, or Val for the amino acidat position 57;Tyr, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Gln, Arg, Ser, Thr,Val, or Trp for the amino acid at position 59;Ser, Ala, Phe, His, Lys, Leu, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyrfor the amino acid at position 61;Trp, Ala, Asp, Glu, Phe, or Gly for the amino acid at position 62;Gly, Ala, Asp, Glu, Phe, Ile, Lys, Leu, Asn, Gln, Arg, Thr, Val, or Trpfor the amino acid at position 65;Arg, Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Asn, Gln, Ser, Thr, Val,Trp, or Tyr for the amino acid at position 96;Phe, Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Asn, Gln, Arg, Ser, Thr,Val, Trp, or Tyr for the amino acid at position 97;Val, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg,Ser, or Thr for the amino acid at position 98;Tyr or Phe for the amino acid at position 100;Thr, Ser, or Val for the amino acid at position 100a;Asp, Ala, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Gln, Arg, Ser, Thr,Val, Trp, or Tyr for the amino acid at position 101; orPro, Asp, or Asn for the amino acid at position 102.

A non-limiting embodiment of the aforementioned flexible residues is anamino acid contained in the light chain variable region, which is anyof:

Trp, Ala, Phe, His, Lys, Asn, Ser, Thr, Val, or Tyr for the amino acidat position 28;Asn, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid at position 29;Tyr, Ala, Asp, Phe, Gly, or His for the amino acid at position 32;Ala, Asp, Glu, Phe, Gly, His, Leu, Gln, Arg, Ser, Thr, Val, or Tyr forthe amino acid at position 93;Asn, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid at position 94;Ser, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg,Thr, Val, Trp, or Tyr for the amino acid at position 95;Gly, Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Val,Trp, or Tyr for the amino acid at position 95a;Trp, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg,Ser, Thr, Val, or Tyr for the amino acid at position 95b; orTyr, Phe, His, Lys, Leu, Asn, or Val for the amino acid at position 95c.

In an embodiment of the present invention, when the small moleculecompound is adenosine diphosphate or adenosine triphosphate, one canidentify the flexible residues and the amino acids that those residuescan be substituted with for library production by crystal structureanalysis of complexes formed by an antibody and adenosine diphosphate oradenosine triphosphate, and introduction of mutations and modeling basedon the crystal structure of a complex formed by an antibody andadenosine which is an analogous compound. For example, residues of theantibody that are not involved in the binding to adenosine diphosphateor adenosine triphosphate can be identified by modeling using crystalstructure analysis of complexes formed by an antibody and adenosinediphosphate or adenosine triphosphate. One can select amino acids thatcan maintain an appropriate level of binding to the compounds even whenthe residues that have been identified as not being involved in thebinding to adenosine diphosphate or adenosine triphosphate aresubstituted with those amino acids. Accordingly, it is possible todesign a library to have selected amino acids at the identifiedresidues. In this case, one can design a library mainly comprising aplurality of antigen-binding molecules that is an assembly ofantigen-binding molecules in which residues identified as not beinginvolved in the binding to adenosine diphosphate or adenosinetriphosphate have been substituted with amino acids that are differentfrom one another. That is, combining each of the flexible residues thathave been substituted with mutually different amino acids can providesequence diversity in antigen-binding molecules containing the flexibleresidues.

Antigen-binding molecules may be designed to include residues wherein atleast one of the residues that are identified to be involved in thebinding to adenosine diphosphate or to adenosine triphosphate becomesany residue selected from that residue and residues different from thatresidue. In a non-limiting embodiment, amino acids that are identifiedas being involved in the binding to the ribose moiety and adenine ringmoiety of adenosine diphosphate or adenosine triphosphate are similar tothose for adenosine, and examples may include any one or more aminoacids selected from amino acids at positions A33, I50, G52, S56, T57,W58, G99, Y100, and T100a (Kabat numbering) in the H chain and atpositions Y95c and N96 (Kabat numbering) in the L chain. For the aminoacids identified as being involved in the binding to the phosphatemoiety of adenosine diphosphate or adenosine triphosphate, one canpredict modifications that can enhance binding to adenosine diphosphateor adenosine triphosphate through examinations based on crystalstructures similar to those described above.

Herein, “flexible residues” refers to amino acid residue variationspresent at hypervariable amino acid positions of light-chain andheavy-chain variable regions at which several different amino acidsexist, when the amino acid sequences of known and/or native antibodiesor antigen-binding domains are compared. The hypervariable positions aregenerally located in the CDR regions. In an embodiment, the dataprovided by Kabat, Sequences of Proteins of Immunological Interest(National Institute of Health Bethesda Md., 1987 and 1991) is useful fordetermining the hypervariable positions in known and/or nativeantibodies. Furthermore, databases on the Internet(http://vbase.mrc-cpe.cam.ac.uk/, andhttp://www.bioinf.org.uk/abs/index.html) provide many collectedsequences of human light chains and heavy chains, and their locations.The information of those sequences and locations is useful fordetermining the hypervariable positions in the present invention.According to the present invention, when a certain amino acid positionhas preferably about 2 to about 20, preferably about 3 to about 19,preferably about 4 to about 18, preferably 5 to 17, preferably 6 to 16,preferably 7 to 15, preferably 8 to 14, preferably 9 to 13, andpreferably 10 to 12 possible amino acid residue variants, the positioncan be considered to be hypervariable. In some embodiments, a certainamino acid position may have preferably at least about 2, preferably atleast about 4, preferably at least about 6, preferably at least about 8,preferably about 10, and preferably about 12 possible amino acid residuevariants.

A library of the present invention that contains a plurality ofantigen-binding molecules having different sequences from one anothercan be constructed by combining heavy chain variable regions produced asa randomized variable region sequence library with the aforementionedlight chain variable regions introduced with at least one amino acidresidue that changes the antigen-binding activity of the antigen-bindingdomains depending on the presence or absence of small molecules.Similarly, a library of the present invention that contains a pluralityof antigen-binding molecules having different sequences from one anothercan also be produced by combining with the heavy-chain variable regionsintroduced with at least one amino acid residue that changes theantigen-binding activity of the antigen-binding domains depending on thepresence or absence of small molecules, and having the other amino acidresidues designed as flexible residues.

When heavy chain variable regions produced as a randomized variableregion sequence library, and light chain variable regions into which atleast one amino acid residue that alters the antigen-binding activity ofan antigen-binding molecule depending on the concentration of the smallmolecule compound has been introduced, are combined as described above,the sequences of the light chain variable regions can be designed tocontain flexible residues in the same manner as described above. Thenumber and position of such flexible residues are not particularlylimited to particular embodiments as long as the antigen-bindingactivity of antigen-binding molecules of the present invention variesdepending on the presence or absence of adenosine and/or ATP.Specifically, the CDR sequences and/or FR sequences of heavy chainand/or light chain can contain one or more flexible residues.

The preferred heavy chain variable regions to be combined include, forexample, randomized variable region libraries. Known methods arecombined as appropriate to produce a randomized variable region library.In a non-limiting embodiment of the present invention, an immune libraryconstructed based on antibody genes derived from lymphocytes of animalsimmunized with a specific antigen, patients with infections, personswith an elevated antibody titer in blood as a result of vaccination,cancer patients, or auto immune disease patients, may be preferably usedas a randomized variable region library.

In another non-limiting embodiment of the present invention, a syntheticlibrary produced by replacing the CDR sequences of V genes in genomicDNA or functional reshaped V genes with a set of syntheticoligonucleotides containing sequences encoding codon sets of anappropriate length can also be preferably used as a randomized variableregion library. In this case, since sequence diversity is observed inthe heavy chain CDR3 sequence, it is also possible to replace the CDR3sequence only. A criterion of giving rise to diversity in amino acids inthe variable region of an antigen-binding molecule is that diversity isgiven to amino acid residues at surface-exposed positions in theantigen-binding molecule. The surface-exposed position refers to aposition that is considered to be able to be exposed on the surfaceand/or contacted with an antigen, based on structure, ensemble ofstructures, and/or modeled structure of an antigen-binding molecule. Ingeneral, such positions are CDRs. Preferably, surface-exposed positionsare determined using coordinates from a three-dimensional model of anantigen-binding molecule using a computer program such as the InsightIIprogram (Accelrys). Surface-exposed positions can be determined usingalgorithms known in the art (for example, Lee and Richards (J. Mol.Biol. (1971) 55, 379-400); Connolly (J. Appl. Cryst. (1983) 16,548-558)). Determination of surface-exposed positions can be performedusing software suitable for protein modeling and three-dimensionalstructural information obtained from an antibody. Software that can beused for these purposes preferably includes SYBYL Biopolymer Modulesoftware (Tripos Associates). Generally or preferably, when an algorithmrequires a user input size parameter, the “size” of a probe which isused in the calculation is set at about 1.4 Angstrom or smaller inradius. Furthermore, methods for determining surface-exposed regions andareas using software for personal computers are described by Pacios(Comput. Chem. (1994) 18 (4), 377-386; J. Mol. Model. (1995) 1, 46-53).

Furthermore, in a non-limiting embodiment of the present invention,amino acids of the variable region including the CDR region and/or theframework region may be altered appropriately to improve antibodystability. In a non-limiting embodiment, examples of such amino acidsmay include the amino acids of positions 1, 5, 10, 30, 48, and 58. Morespecifically, examples may include Gln at position 1, Gln at position 5,Asp at position 10, Asn at position 30, Leu at position 48, and Asn atposition 58. For the improvement of antibody stability, these aminoacids can be substituted with corresponding amino acids contained in agerm-line sequence. In a non-limiting embodiment, an example of such agerm line sequence may be the VH3-21 sequence. In this case, Gln ofposition 1 may be substituted with Glu, Gln of position 5 may besubstituted with Val, Asp of position 10 may be substituted with Gly,Asn of position 30 may be substituted with Ser, Leu of position 48 maybe substituted with Val, and Asn of position 58 may be substituted withTyr.

In another non-limiting embodiment of the present invention, a naivelibrary which is constructed from antibody genes derived fromlymphocytes of healthy individuals and consists of naive sequences whichare antibody sequences that do not have bias in their repertoire, canalso be particularly preferably used as a randomized variable regionlibrary (Gejima et al. (Human Antibodies (2002) 11, 121-129); Cardoso etal. (Scand. J. Immunol. (2000) 51, 337-344)). Herein, “an amino acidsequence comprising a naive sequence” refers to an amino acid sequenceobtained from such a naive library.

Fc Region

An Fc region contains an amino acid sequence derived from the heavychain constant region of an antibody. An Fc region is a portion of theantibody heavy chain constant region that includes the N terminal end ofthe hinge region, which is the papain cleavage site, at an amino acidaround position 216 (indicated by EU numbering), and the hinge, CH2, andCH3 domains. Fc regions can be obtained from human IgG1; however, theyare not limited to any specific IgG subclass. Preferred examples of theFc regions include Fc regions having FcRn-binding activity in an acidicpH range as described below. Preferred examples of the Fc regionsinclude Fc regions having Fcγ receptor-binding activity as describedbelow. In a non-limiting embodiment, examples of such Fc regions includethe Fc regions of human IgG1 (SEQ ID NO: 5), IgG2 (SEQ ID NO: 6), IgG3(SEQ ID NO: 7), or IgG4 (SEQ ID NO: 8).

Fcγ Receptor (FcγR)

“Fcγ receptor” (also called “FcγR”) refers to a receptor capable ofbinding to the Fc region of monoclonal IgG1, IgG2, IgG3, or IgG4antibodies; and means all members belonging to the family of proteinssubstantially encoded by Fcγ receptor genes. In humans, the familyincludes FcγRI (CD64) including isoforms FcγRIa, FcγRIb, and FcγRIc;FcγRII (CD32) including isoforms FcγRIIa (including allotype H131 andR131, i.e., FcγRIIa(H) and FcγRIIa(R)), FcγRIIb (including FcγRIIb-1 andFcγRIIb-2), and FcγRIIc; and FcγRIII (CD16) including isoform FcγRIIIa(including allotype V158 and F158, i.e., FcγRIIIa(V) and FcγRIIIa(F))and FcγRIIIb (including allotype FcγRIIIb-NA1 and FcγRIIIb-NA2); as wellas all unidentified human FcγRs, FcγR isoforms, and allotypes thereof;but the family is not limited to these examples. Without being limitedthereto, FcγRs include those derived from humans, mice, rats, rabbits,and monkeys. FcγRs may be derived from any organism. Mouse FcγRs includeFcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (FcγRIV,CD16-2), as well as all unidentified mouse FcγRs, FcγR isoforms, andallotypes thereof, but they are not limited to these examples. Preferredexamples of such Fcγ receptors include, human FcγRI (CD64), FcγRIIa(CD32), FcγRIIb (CD32), FcγRIIIa (CD16), and/or FcγRIIIb (CD16). Thepolynucleotide sequence and amino acid sequence of human FcγRI are shownin SEQ ID NOs: 9 (NM_000566.3) and 10 (NP_000557.1), respectively; thepolynucleotide sequence and amino acid sequence of human FcγRIIa(allotype H131) are shown in SEQ ID NOs: 11 (BC020823.1) and 12(AAH20823.1), respectively (allotype R131 is a sequence in which theamino acid at position 166 of SEQ ID NO: 12 is substituted with Arg);the polynucleotide sequence and amino acid sequence of FcγIIb are shownin SEQ ID NOs: 13 (BC146678.1) and 14 (AAI46679.1), respectively; thepolynucleotide sequence and amino acid sequence of FcγRIIIa are shown inSEQ ID NOs: 15 (BC033678.1) and 16 (AAH33678.1), respectively; and thepolynucleotide sequence and amino acid sequence of FcγRIIIb are shown inSEQ ID NOs: 17 (BC128562.1) and 18 (AAI28563.1), respectively (RefSeqaccession number or such is shown in parentheses). Whether an Fcγreceptor has binding activity to the Fc region of a monoclonal IgG1,IgG2, IgG3, or IgG4 antibody can be assessed by ALPHA (AmplifiedLuminescent Proximity Homogeneous Assay) screen, surface plasmonresonance (SPR)-based BIACORE methods, and others (Proc. Natl. Acad.Sci. USA (2006) 103(11), 4005-4010), in addition to the above-describedFACS and ELISA formats.

In FcγRI (CD64) including FcγRIa, FcγRIb, and FcγRIc, and FcγRIII (CD16)including isoforms FcγRIIIa (including allotypes V158 and F158) andFcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), a chainthat binds to the Fc region of IgG is associated with common γ chainhaving ITAM responsible for transduction of intracellular activationsignal. Meanwhile, the cytoplasmic domain of FcγRII (CD32) includingisoforms FcγRIIa (including allotypes H131 and R131) and FcγRIIccontains ITAM. These receptors are expressed on many immune cells suchas macrophages, mast cells, and antigen-presenting cells. The activationsignal transduced upon binding of these receptors to the Fc region ofIgG results in enhancement of the phagocytic activity of macrophages,inflammatory cytokine production, mast cell degranulation, and theenhanced function of antigen-presenting cells. Fcγ receptors having theability to transduce the activation signal as described above are hereinreferred to as activating Fcγ receptors.

Meanwhile, the intracytoplasmic domain of FcγRIIb (including FcγRIIb-1and FcγRIIb-2) contains ITIM responsible for transduction of inhibitorysignals. The crosslinking between FcγRIIb and B cell receptor (BCR) on Bcells suppresses the activation signal from BCR, which results insuppression of antibody production via BCR. The crosslinking of FcγRIIIand FcγRIIb on macrophages suppresses the phagocytic activity andinflammatory cytokine production. Fcγ receptors having the ability totransduce the inhibitory signal as described above are herein referredto as inhibitory Fcγ receptor.

FcγR-Binding Activity of Fc Region

As mentioned above, Fc regions having an Fcγ receptor-binding activityare examples of Fc regions comprised in the antigen-binding molecules ofthe present invention. A non-limiting embodiment of such an Fc regionincludes the Fc region of human IgG1 (SEQ ID NO: 5), IgG2 (SEQ ID NO:6), IgG3 (SEQ ID NO: 7), or IgG4 (SEQ ID NO: 8). Whether an Fcγ receptorhas binding activity to the Fc region of a monoclonal IgG1, IgG2, IgG3,or IgG4 antibody can be assessed by ALPHA screen (Amplified LuminescentProximity Homogeneous Assay), surface plasmon resonance (SPR)-basedBIACORE method, and others (Proc. Natl. Acad. Sci. U.S.A. (2006)103(11), 4005-4010), in addition to the above-described FACS and ELISAformats.

ALPHA screen is performed by the ALPHA technology based on the principledescribed below using two types of beads: donor and acceptor beads. Aluminescent signal is detected only when molecules linked to the donorbeads interact biologically with molecules linked to the acceptor beadsand when the two beads are located in close proximity. Excited by laserbeam, the photosensitizer in a donor bead converts oxygen around thebead into excited singlet oxygen. When the singlet oxygen diffusesaround the donor beads and reaches the acceptor beads located in closeproximity, a chemiluminescent reaction within the acceptor beads isinduced. This reaction ultimately results in light emission. Ifmolecules linked to the donor beads do not interact with moleculeslinked to the acceptor beads, the singlet oxygen produced by donor beadsdo not reach the acceptor beads and chemiluminescent reaction does notoccur.

For example, a biotin-labeled antigen-binding molecule comprising Fcregion is immobilized to the donor beads and glutathione S-transferase(GST)-tagged Fcγ receptor is immobilized to the acceptor beads. In theabsence of an antigen-binding molecule comprising a competitive Fcregion variant, Fcγ receptor interacts with an antigen-binding moleculecomprising a native Fc region, inducing a signal of 520 to 620 nm as aresult. The antigen-binding molecule having a non-tagged Fc regionvariant competes with the antigen-binding molecule comprising a nativeFc region for the interaction with Fcγ receptor. The relative bindingaffinity can be determined by quantifying the reduction of fluorescenceas a result of competition. Methods for biotinylating theantigen-binding molecules such as antibodies using Sulfo-NHS-biotin orthe like are known. Appropriate methods for adding the GST tag to an Fcγreceptor include methods that involve fusing polypeptides encoding Fcγand GST in-frame, expressing the fused gene using cells introduced witha vector to which the gene is operably linked, and then purifying usinga glutathione column. The induced signal can be preferably analyzed, forexample, by fitting to a one-site competition model based on nonlinearregression analysis using software such as GRAPHPAD PRISM (GraphPad; SanDiego).

One of the substances for observing their interaction is immobilized asa ligand onto the gold thin layer of a sensor chip. When light is shedon the rear surface of the sensor chip so that total reflection occursat the interface between the gold thin layer and glass, the intensity ofreflected light is partially reduced at a certain site (SPR signal). Theother substance for observing their interaction is injected as ananalyte onto the surface of the sensor chip. The mass of immobilizedligand molecule increases when the analyte binds to the ligand. Thisalters the refraction index of solvent on the surface of the sensorchip. The change in refraction index causes a positional shift of SPRsignal (conversely, the dissociation shifts the signal back to theoriginal position). In the Biacore system, the amount of shift describedabove (i.e., the change of mass on the sensor chip surface) is plottedon the vertical axis, and thus the change of mass over time is shown asmeasured data (sensorgram). Kinetic parameters (association rateconstant (ka) and dissociation rate constant (kd)) are determined fromthe curve of sensorgram, and affinity (KD) is determined from the ratiobetween these constants. Inhibition assay is preferably used in theBIACORE methods. Examples of such inhibition assay are described inProc. Natl. Acad. Sci. U.S.A. (2006) 103(11), 4005-4010.

Fc Regions with Altered Fcγ Receptor (FcγR) Binding

In addition to the Fc region of human IgG1 (SEQ ID NO: 5), IgG2 (SEQ IDNO: 6), IgG3 (SEQ ID NO: 7), or IgG4 (SEQ ID NO: 8), an Fc region withaltered FcγR binding, which has a higher Fcγ receptor-binding activitythan an Fc region of a native human IgG may be appropriately used as anFc region included in the present invention. Herein, “Fc region of anative human IgG” refers to an Fc region in which the sugar chain bondedto position 297 (EU numbering) of the Fc region of human IgG1, IgG2,IgG3, or IgG4 shown in SEQ ID NOs: 5, 6, 7, or 8 is a fucose-containingsugar chain. Such Fc regions with altered FcγR binding may be producedby altering amino acids of the Fc region of a native human IgG. Whetherthe FcγR-binding activity of an Fc region with altered FcγR binding ishigher than that of an Fc region of a native human IgG can be determinedappropriately using methods described in the abovementioned section onbinding activity.

In the present invention, “alteration of amino acids” or “amino acidalteration” of an Fc region includes alteration into an amino acidsequence which is different from that of the starting Fc region. Thestarting Fc region may be any Fc region, as long as a variant modifiedfrom the starting Fc region can bind to human Fcγ receptor in a neutralpH range. Furthermore, an Fc region altered from a starting Fc regionwhich had been already altered can also be used preferably as an Fcregion of the present invention. The “starting Fc region” can refer tothe polypeptide itself, a composition comprising the starting Fc region,or an amino acid sequence encoding the starting Fc region. Starting Fcregions can comprise known Fc regions produced via recombinationdescribed briefly in the section “Antibodies”. The origin of starting Fcregions is not limited, and they may be obtained from human or anynonhuman organisms. Such organisms preferably include mice, rats, guineapigs, hamsters, gerbils, cats, rabbits, dogs, goats, sheep, bovines,horses, camels and organisms selected from nonhuman primates. In anotherembodiment, starting Fc regions can also be obtained from cynomolgusmonkeys, marmosets, rhesus monkeys, chimpanzees, or humans. Starting Fcregions can be obtained preferably from human IgG1; however, they arenot limited to any particular IgG class. This means that an Fc region ofhuman IgG1, IgG2, IgG3, or IgG4 can be used appropriately as a startingFc region, and herein also means that an Fc region of an arbitrary IgGclass or subclass derived from any organisms described above can bepreferably used as a starting Fc region. Examples of native IgG variantsor altered forms are described in published documents (Curr. Opin.Biotechnol. (2009) 20 (6): 685-91; Curr. Opin. Immunol. (2008) 20 (4),460-470; Protein Eng. Des. Sel. (2010) 23 (4): 195-202; InternationalPublication Nos. WO 2009/086320, WO 2008/092117, WO 2007/041635, and WO2006/105338); however, they are not limited to the examples.

Examples of alterations include those with one or more mutations, forexample, mutations by substitution of different amino acid residues foramino acids of starting Fc regions, by insertion of one or more aminoacid residues into starting Fc regions, or by deletion of one or moreamino acids from starting Fc region. Preferably, the amino acidsequences of altered Fc regions comprise at least a part of the aminoacid sequence of a non-native Fc region. Such variants necessarily havesequence identity or similarity less than 100% to their starting Fcregion. In a preferred embodiment, the variants have amino acid sequenceidentity or similarity about 75% to less than 100%, more preferablyabout 80% to less than 100%, even more preferably about 85% to less than100%, still more preferably about 90% to less than 100%, and yet morepreferably about 95% to less than 100% to the amino acid sequence oftheir starting Fc region. In a non-limiting embodiment of the presentinvention, at least one amino acid is different between an FcγR-bindingaltered Fc region of the present invention and its starting Fc region.Amino acid difference between an FcγR-binding altered Fc region of thepresent invention and its starting Fc region can also be preferablyspecified based on the specific amino acid differences at theabove-described specific amino acid positions by EU numbering. Examplesof methods of preparing such variants are shown in the section“Alteration of amino acids”.

Included in the antigen-binding molecules of the present invention, anFc region with altered FcγR binding, which has a higher Fcγreceptor-binding activity than that of an Fc region of a native humanIgG, (an FcγR binding-altered Fc region) may be obtained by any method.Specifically, the Fc region with altered FcγR binding may be obtained byaltering amino acids of an IgG-type human immunoglobulin used as astarting Fc region. Preferred Fc regions of the IgG-type immunoglobulinsfor alteration include, for example, those of human IgGs shown in SEQ IDNOs: 5, 6, 7, or 8 (IgG1, IgG2, IgG3, or IgG4, respectively, andvariants thereof).

Amino acids of any positions may be altered into other amino acids, aslong as the binding activity toward the Fcγ receptor is higher than thatof the Fc region of a native human IgG. When the antigen-bindingmolecule contains a human IgG1 Fc region as the human Fc region, itpreferably contains an alteration that yields the effect of a higher Fcγreceptor-binding activity than that of the Fc region of a native humanIgG, in which the sugar chain bound at position 297 (EU numbering) is afucose-containing sugar chain. Such amino acid alterations have beenreported, for example, in international publications such asWO2007/024249, WO2007/021841, WO2006/031370, WO2000/042072,WO2004/029207, WO2004/099249, WO2006/105338, WO2007/041635,WO2008/092117, WO2005/070963, WO2006/020114, WO2006/116260, andWO2006/023403.

For the pH conditions to measure the binding activity of the Fcγreceptor binding domain and the Fcγ receptor contained in theantigen-binding molecule of the present invention, conditions in anacidic pH range or in a neutral pH range may be suitably used. Theacidic pH range or neutral pH range, as a condition to measure thebinding activity of the Fcγ receptor binding domain and the Fcγ receptorcontained in the antigen-binding molecule of the present invention,generally indicates pH 5.8 to pH 8.0. Preferably, it is a rangeindicated with arbitrary pH values between pH 6.0 and pH 7.4; andpreferably, it is selected from pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4,pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3,and pH 7.4; and particularly preferably, it is pH 6.15 to 7.4, which isclose to the pH of cancer tissues (Vaupel et al., Cancer Res. (1989) 49,6449-6665). With regard to the temperature used as a measurementcondition, the binding affinity between an Fcγ receptor binding domainand a human Fcγ receptor can be evaluated at any temperature between 10°C. and 50° C. Preferably, a temperature between 15° C. and 40° C. isused to determine the binding affinity between a human Fcγ receptorbinding domain and Fcγ receptor. More preferably, any temperaturebetween 20° C. and 35° C., such as any single temperature from 20° C.,21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C.,30° C., 31° C., 32° C., 33° C., 34° C., and 35° C., can be similarlyused to determine the binding affinity between an Fcγ receptor bindingdomain and an Fcγ receptor. A temperature of 25° C. is a non-limitingexample in an embodiment of the present invention.

Herein, “Fc region with altered FcγR binding has a higher Fcγreceptor-binding activity than the native Fc region” means that thehuman Fcγ receptor-binding activity of the Fc region with altered FcγRbinding toward any of the human Fcγ receptors of FcγRI, FcγRIIa,FcγRIIb, FcγRIIIa, and/or FcγRIIIb is higher than the binding activityof the native Fc region toward these human Fcγ receptors. For example,it means that based on an above-described analytical method, incomparison to the binding activity of an antigen-binding moleculecontaining a native human IgG Fc region as a control, the bindingactivity of the antigen-binding molecule comprising an Fc region withaltered FcγR binding is 105% or more, preferably 110% or more, 115% ormore, 120% or more, 125% or more, particularly preferably 130% or more,135% or more, 140% or more, 145% or more, 150% or more, 155% or more,160% or more, 165% or more, 170% or more, 175% or more, 180% or more,185% or more, 190% or more, 195% or more, 2-fold or more, 2.5-fold ormore, 3-fold or more, 3.5-fold or more, 4-fold or more, 4.5-fold ormore, 5-fold or more, 7.5-fold or more, 10-fold or more, 20-fold ormore, 30-fold or more, 40-fold or more, 50-fold or more, 60-fold ormore, 70-fold or more, 80-fold or more, 90-fold or more, or 100-fold ormore. The starting Fc region may be used as a native Fc region, andnative Fc regions of antibodies of the same subclass may also be used.

In the present invention, an Fc region of a native human IgG in whichthe sugar chain bonded to the amino acid at position 297 (EU numbering)is a fucose-containing sugar chain, is suitably used as a native Fcregion of human IgG to be used as a control. Whether or not the sugarchain bonded to the amino acid at position 297 (EU numbering) is afucose-containing sugar chain can be determined using a known technique(Non-fucosylated therapeutic antibodies as next-generation therapeuticantibodies. Satoh M, Iida S, Shitara K., Expert Opin. Biol. Ther. (2006)6 (11), 1161-1173). For example, it is possible to determine whether ornot the sugar chain bonded to the native human IgG Fc region is afucose-containing sugar chain by a method such as the one below. Sugarchain is dissociated from a native human IgG to be tested, by reactingthe test native human IgG with N-Glycosidase F (Roche diagnostics)(Weitzhandler et al. (J. Pharma. Sciences (1994) 83, 12, 1670-1675)).Next, a dried concentrate of a reaction solution from which protein hasbeen removed by reaction with ethanol (Schenk et al. (J. Clin.Investigation (2001) 108 (11) 1687-1695)) is fluorescently labeled with2-aminopyridine (Bigge et al. (Anal. Biochem. (1995) 230 (2) 229-238)).Reagents are removed by solid extraction using a cellulose cartridge,and the fluorescently labeled 2-AB-modified sugar chain is analyzed bynormal-phase chromatography. It is possible to determine whether or notthe sugar chain bonded to the native Fc region of a human IgG is afucose-containing sugar chain by observing the detected chromatogrampeaks.

As an antigen-binding molecule containing a native Fc region of anantibody of the same subclass, which is to be used as a control, anantigen-binding molecule having an Fc region of a monoclonal IgGantibody may be suitably used. The structures of the Fc regions aredescribed in SEQ ID NO: 5 (A is added to the N terminus of DatabaseAccession No. AAC82527.1), SEQ ID NO: 6 (A is added to the N terminus ofDatabase Accession No. AAB59393.1), SEQ ID NO: 7 (Database Accession No.CAA27268.1), and SEQ ID NO: 8 (A is added to the N terminus of DatabaseAccession No. AAB59394.1). Further, when an antigen-binding moleculecontaining an Fc region of a particular antibody isotype is used as thetest substance, the effect of the antigen-binding molecule containingthe test Fc region on Fcγ receptor-binding activity is tested by usingas a control an antigen-binding molecule having an Fc region of amonoclonal IgG antibody of that particular isotype. In this way,antigen-binding molecules containing an Fc region of which Fcγreceptor-binding activity is demonstrated to be high are suitablyselected.

Fc Regions Having a Selective Binding Activity Toward an Fcγ Receptor

Examples of Fcγ receptor binding domains suitable for use in the presentinvention include Fcγ receptor binding domains having a higher bindingactivity to a particular Fcγ receptor than to other Fcγ receptors (Fcγreceptor binding domains having a selective binding activity to an Fcγreceptor). When an antibody is used as the antigen-binding molecule(when an Fc region is used as the Fcγ receptor binding domain), a singleantibody molecule can only bind to a single Fcγ receptor molecule.Therefore, a single antigen-binding molecule cannot bind to otheractivating FcγRs in an inhibitory Fcγ receptor-bound state, and cannotbind to other activating Fcγ receptors or inhibitory Fcγ receptors in anactivating Fcγ receptor-bound state.

Fc Regions with a Higher Binding Activity Toward an Activating FcγReceptor than the Binding Activity Toward an Inhibitory Fcγ Receptor

As described above, preferable activating Fcγ receptors include FcγRI(CD64) including FcγRIa, FcγRIb, and FcγRIc; FcγRIIa; and FcγRIII (CD16)including FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb(including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2). Meanwhile,preferred examples of inhibitory Fcγ receptors include FcγRIIb(including FcγRIIb-1 and FcγRIIb-2).

Herein, an example of a case where the binding activity toward a certainFcγ receptor is higher than the binding activity toward another Fcγreceptor is the case where the binding activity toward an activating Fcγreceptor is higher than the binding activity toward an inhibitory Fcγreceptor. In this case, the binding activity of the Fc region toward anyof the human Fcγ receptors of FcγRIa, FcγRIIa, FcγRIIIa, and/or FcγRIIIbis said to be higher than the binding activity toward FcγRIIb. Forexample, this means that, based on an above-described analytical method,the binding activity of an antigen-binding molecule containing the Fcregion toward any of the human Fcγ receptors, FcγRIa, FcγRIIa, FcγRIIIa,and/or FcγRIIIb, is 105% or more, preferably 110% or more, 120% or more,130% or more, 140% or more, particularly preferably 150% or more, 160%or more, 170% or more, 180% or more, 190% or more, 200% or more, 250% ormore, 300% or more, 350% or more, 400% or more, 450% or more, 500% ormore, 750% or more, 10-fold or more, 20-fold or more, 30-fold or more,40-fold or more, 50-fold or more, 60-fold, 70-fold, 80-fold, 90-fold, or100-fold or more as compared with the binding activity toward FcγRIIb.The Fc region with a higher binding activity toward activating Fcγreceptors than to inhibitory Fcγ receptors may be favorably included inantigen-binding molecules of the present invention whose antigen-bindingdomain binds to a membrane-type molecule. IgG1 antibodies containingsuch Fc regions are known to enhance the ADCC activity mentioned below.Therefore, antigen-binding molecules containing the Fc-region are alsouseful as antigen-binding molecules to be included in the pharmaceuticalcompositions of the present invention.

In a non-limiting embodiment of the present invention, examples of theFc region with a higher binding activity toward activating Fcγ receptorsthan to inhibitory Fcγ receptors (or having a selective binding activitytoward inhibitory Fcγ receptors) preferably include Fc regions in whichat least one or more amino acids selected from the group consisting ofamino acids at positions 221, 222, 223, 224, 225, 227, 228, 230, 231,232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246,247, 249, 250, 251, 254, 255, 256, 258, 260, 262, 263, 264, 265, 266,267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281,282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297,298, 299, 300, 301, 302, 303, 304, 305, 311, 313, 315, 317, 318, 320,322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335,336, 337, 339, 376, 377, 378, 379, 380, 382, 385, 392, 396, 421, 427,428, 429, 434, 436, and 440 indicated by EU numbering mentioned above,have been altered to amino acids different from those of the native Fcregion.

Fc Regions Whose Binding Activity Toward an Inhibitory Fcγ Receptor isHigher than the Binding Activity Toward an Activating Fcγ Receptor

Herein, an example of a case where the binding activity toward a certainFcγ receptor is higher than the binding activity toward another Fcγreceptor is the case where the binding activity toward an inhibitory Fcγreceptor is higher than the binding activity toward an activating Fcγreceptor. In this case, the binding activity of the Fc region towardFcγRIIb is said to be higher than the binding activity toward any of thehuman Fcγ receptors of FcγRIa, FcγRIIa, FcγRIIIa, and/or FcγRIIIb. Forexample, this means that, based on an above-described analytical method,the binding activity of an antigen-binding molecule containing the Fcregion toward FcγRIIb is 105% or more, preferably 110% or more, 120% ormore, 130% or more, 140% or more, particularly preferably 150% or more,160% or more, 170% or more, 180% or more, 190% or more, 200% or more,250% or more, 300% or more, 350% or more, 400% or more, 450% or more,500% or more, 750% or more, 10-fold or more, 20-fold or more, 30-fold ormore, 40-fold or more, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or100-fold or more as compared with the binding activity toward any of thehuman Fcγ receptors of FcγRIa, FcγRIIa, FcγRIIIa, and/or FcγRIIIb. TheFc region with a higher binding activity toward inhibitory Fcγ receptorsthan to activating Fcγ receptors may be favorably included inantigen-binding molecules of the present invention whose antigen-bindingdomain binds to a soluble molecule.

In a non-limiting embodiment of the present invention, examples of theFc region with a higher binding activity toward inhibitory Fcγ receptorsthan to activating Fcγ receptors (or having a selective binding activitytoward inhibitory Fcγ receptors) preferably include Fc regions in which,of the amino acids of the above Fc region, the amino acids at 238 and328 indicated by EU numbering are altered to amino acids different fromthose of the native Fc region.

In a non-limiting embodiment of the present invention, examples of theFc region with a higher binding activity toward inhibitory Fcγ receptorsthan to activating Fcγ receptors (or having a selective binding activitytoward inhibitory Fcγ receptors) preferably include Fc regions alteredat any one or more of the amino acids in the above Fc region asindicated by EU numbering: the amino acid at position 238 (indicated byEU numbering) is altered into Asp; and the amino acid at position 328(indicated by EU numbering) is altered into Glu. Furthermore, as the Fcregions having a selective binding activity toward inhibitory Fcγreceptors, the Fc regions or alterations described in US 2009/0136485can be suitably selected.

In another non-limiting embodiment of the present invention, preferredexamples include Fc regions altered at any one or more of the aminoacids in the above Fc region as indicated by EU numbering: the aminoacid at position 238 (indicated by EU numbering) to Asp; and the aminoacid at position 328 (indicated by EU numbering) to Glu.

In still another non-limiting embodiment of the present invention,preferred examples include Fc regions that have one or more of thealterations exemplified in PCT/JP2012/054624: substitution of Pro atposition 238 (indicated by EU numbering) with Asp; alteration of theamino acid at position 237 (indicated by EU numbering) to Trp;alteration of the amino acid at position 237 (indicated by EU numbering)to Phe; alteration of the amino acid at position 267 (indicated by EUnumbering) to Val; alteration of the amino acid at position 267(indicated by EU numbering) to Gln; alteration of the amino acid atposition 268 (indicated by EU numbering) to Asn; alteration of the aminoacid at position 271 (indicated by EU numbering) to Gly; alteration ofthe amino acid at position 326 (indicated by EU numbering) to Leu;alteration of the amino acid at position 326 (indicated by EU numbering)to Gln; alteration of the amino acid at position 326 (indicated by EUnumbering) to Glu; alteration of the amino acid at position 326(indicated by EU numbering) to Met; alteration of the amino acid atposition 239 (indicated by EU numbering) to Asp; alteration of the aminoacid at position 267 (indicated by EU numbering) to Ala; alteration ofthe amino acid at position 234 (indicated by EU numbering) to Trp;alteration of the amino acid at position 234 (indicated by EU numbering)to Tyr; alteration of the amino acid at position 237 (indicated by EUnumbering) to Ala; alteration of the amino acid at position 237(indicated by EU numbering) to Asp; alteration of the amino acid atposition 237 (indicated by EU numbering) to Glu; alteration of the aminoacid at position 237 (indicated by EU numbering) to Leu; alteration ofthe amino acid at position 237 (indicated by EU numbering) to Met;alteration of the amino acid at position 237 (indicated by EU numbering)to Tyr; alteration of the amino acid at position 330 (indicated by EUnumbering) to Lys; alteration of the amino acid at position 330(indicated by EU numbering) to Arg, alteration of the amino acid atposition 233 (indicated by EU numbering) to Asp, alteration of the aminoacid at position 268 (indicated by EU numbering) to Asp, alteration ofthe amino acid at position 268 (indicated by EU numbering) to Glu,alteration of the amino acid at position 326 (indicated by EU numbering)to Asp, alteration of the amino acid at position 326 (indicated by EUnumbering) to Ser, alteration of the amino acid at position 326(indicated by EU numbering) to Thr, alteration of the amino acid atposition 323 (indicated by EU numbering) to Ile, alteration of the aminoacid at position 323 (indicated by EU numbering) to Leu, alteration ofthe amino acid at position 323 (indicated by EU numbering) to Met,alteration of the amino acid at position 296 (indicated by EU numbering)to Asp, alteration of the amino acid at position 326 (indicated by EUnumbering) to Ala, alteration of the amino acid at position 326(indicated by EU numbering) to Asn, and alteration of the amino acid atposition 330 (indicated by EU numbering) to Met.

Fc Regions with Modified Sugar Chains

Fc regions contained in the antigen-binding molecules provided by thepresent invention may include Fc regions that have been modified so thatthe composition of the sugar-chain-attached Fc regions has a highpercentage of fucose-deficient sugar-chain-attached Fc regions, or ahigh percentage of bisecting N-acetylglucosamine-added Fc regions.Removal of fucose residue from N-acetylglucosamine at the reducing endof N-glycoside linkage complex sugar chains bonded to the antibody Fcregion is known to enhance the affinity to FcγRIIIa (Non-Patent Document6). It is known that for IgG1 antibodies containing such Fc regions, theADCC activity mentioned below is enhanced; therefore, antigen-bindingmolecules containing such Fc regions are also useful as antigen-bindingmolecules to be contained in pharmaceutical compositions of the presentinvention. Examples of antibodies with fucose residue removed fromN-acetylglucosamine at the reducing end of N-glycoside linkage complexsugar chains bonded to the antibody Fc regions are antibodies such as:

antibodies modified by glycosylation (for example, WO 1999/054342); andantibodies deficient in fucose attached to sugar chains (for example, WO2000/061739, WO 2002/031140, and WO 2006/067913).

More specifically, to produce antibodies deficient in fucose attached tosugar chains (for example, WO 2000/061739, WO 2002/031140, and WO2006/067913) as another non-limiting embodiment of antibodies withfucose residue removed from N-acetylglucosamine at the reducing end ofN-glycoside linkage complex sugar chains bonded to the antibody Fcregions, host cells having a low ability to add fucose to sugar chainsare produced by altering the activity of forming the sugar chainstructure of the polypeptide to be glycosylated. Antibodies that lackfucose in their sugar chains can be collected from culture of the hostcells by expressing a desired antibody gene in the host cells.Non-limiting suitable examples of the activity to form the sugar chainstructure of a polypeptide include the activity of a transporter or anenzyme selected from the group consisting of fucosyltransferase (EC2.4.1.152), fucose transporter (SLC35C1), GMD(GDP-mannose-4,6-dehydratase) (EC 4.2.1.47), Fx(GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase) (EC 1.1.1.271), andGFPP (GDP-β-L-fucose pyrophosphorylase (EC 2.7.7.30). As long as theseenzymes or transporters can exhibit their activities, their structuresare not necessarily specified. Herein, proteins that can exhibit theseactivities are referred to as “functional proteins”. In a non-limitingembodiment, methods for altering these activities include deletion ofthese activities. To produce host cells deficient in these activities,known methods such as a method for destroying the genes of thesefunctional proteins to make them unable to function may be appropriatelyemployed (for example, WO2000/061739, WO2002/031140, and WO2006/067913).Host cells deficient in such activities can be produced, for example, bya method that destroys the genes of these functional proteins endogenousto CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63mouse myeloma cells, PER cells, PER.C6 cells, HEK293 cells, hybridomacells, or such, so that the genes are unable to function.

Antibodies that have a sugar chain containing bisecting GlcNAc(WO2002/079255, etc.) are known. In a non-limiting embodiment, hostcells for expressing a gene that encodes a functional protein havingGnTIII (β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase)(EC 2.4.1.144) activity or GalT (β-1,4-galactosyltransferase) (EC2.4.1.38) activity are produced to prepare antibodies that havebisecting GlcNAc-containing sugar chains. In another suitablenon-limiting embodiment, host cells that co-express, in addition to theaforementioned functional proteins, a gene encoding a functional proteinhaving human ManII (mannosidase II) (3.2.1.114) activity, a geneencoding a functional protein having GnTI(β-1,2-acetylglucosaminyltransferase I) (EC 2.4.1.94) activity, a geneencoding a functional protein having GnTII(β-1,2-acetylglucosaminyltransferase II) (EC 2.4.1.143) activity, a geneencoding a functional protein having ManI (mannosidase) (EC 3.2.1.113)activity, and α-1,6-fucosyl transferase (EC 2.4.1.68), are produced(WO2004/065540).

Antibodies with fucose residue removed from N-acetylglucosamine at thereducing end of N-glycoside linkage complex sugar chains bonded to theantibody Fc regions and antibodies having sugar chains containingbisecting GlcNAc can be produced, respectively, by transfecting anexpression vector containing the antibody gene into host cells with alow ability to add fucose to sugar chains, and into host cells havingthe activity to form bisecting GlcNAc structure-containing sugar chains.Methods for producing these antibodies can be applied to methods forproducing antigen-binding molecules containing altered Fc regions thathave been modified so that the composition of the sugar-chain-attachedFc regions of the present invention has a high percentage offucose-deficient sugar chain-attached Fc regions or a high percentage ofbisecting N-acetylglucosamine-added Fc regions. The composition of thesugar-chain-attached Fc regions contained in the antigen-bindingmolecules of the present invention produced by such production methodscan be assessed by the method described in “Fc regions with altered Fγγreceptor (FcγR) binding” above.

Multispecific Antigen-Binding Molecules or MultiparatopicAntigen-Binding Molecules

An antigen-binding molecule comprising at least two antigen-bindingdomains in which at least one of the antigen-binding domains binds to afirst epitope in an antigen molecule, and at least another one of theantigen-binding domains binds to a second epitope in the antigenmolecule, is called “multispecific antigen-binding molecule” from theviewpoint of its reaction specificity. When two types of antigen-bindingdomains contained in a single antigen-binding molecule allow binding totwo different epitopes by the antigen-binding molecule, this molecule iscalled “bispecific antigen-binding molecule”. When three types ofantigen-binding domains contained in a single antigen-binding moleculeallow binding to three different epitopes by the antigen-bindingmolecule, this antigen-binding molecule is called “trispecificantigen-binding molecule”.

A paratope in the antigen-binding domain that binds to the first epitopein the antigen molecule and a paratope in the antigen-binding domainthat binds to the second epitope which is structurally different fromthe first epitope have different structures. Therefore, anantigen-binding molecule comprising at least two antigen-binding domainsin which at least one of the antigen-binding domains binds to a firstepitope in an antigen molecule, and at least another one of theantigen-binding domains binds to a second epitope in the antigenmolecule, is called “multiparatopic antigen-binding molecule” from theviewpoint of the specificity of its structure. When two types ofantigen-binding domains contained in a single antigen-binding moleculeallow binding to two different epitopes by the antigen-binding molecule,this molecule is called “biparatopic antigen-binding molecule”. Whenthree types of antigen-binding domains contained in a singleantigen-binding molecule allow binding to three different epitopes bythe antigen-binding molecule, this molecule is called “triparatopicantigen-binding molecule”.

Multivalent multispecific or multiparatopic antigen-binding moleculescomprising one or more antigen-binding domains and methods for preparingthem are described in non-patent documents such as Conrath et al., (J.Biol. Chem. (2001) 276 (10) 7346-7350), Muyldermans (Rev. Mol. Biotech.(2001) 74, 277-302), and Kontermann R. E. (2011) Bispecific Antibodies(Springer-Verlag), and in patent documents such as WO1996/034103 andWO1999/023221. Antigen-binding molecules of the present invention can beproduced using multispecific or multiparatopic antigen-bindingmolecules, and their preparation methods described in these documents.

Bispecific Antibodies and Methods for Producing them

In an embodiment, bispecific antibodies and methods for producing themare mentioned below as examples of the aforementioned multispecific ormultiparatopic antigen-binding molecules and methods for preparing them.Bispecific antibodies are antibodies comprising two types of variableregions that bind specifically to different epitopes. IgG-typebispecific antibodies can be secreted from a hybrid hybridoma (quadroma)produced by fusing two types of hybridomas that produce IgG antibodies(Milstein et al., Nature (1983) 305, 537-540).

When a bispecific antibody is produced by using recombination techniquessuch as those described in the above-mentioned section on antibodies,one may adopt a method that introduces genes encoding heavy chainscontaining the two types of variable regions of interest into cells toco-express them. However, even when only the heavy-chain combination isconsidered, such a co-expression method will produce a mixture of (i) acombination of a pair of heavy chains in which one of the heavy chainscontains a variable region that binds to a first epitope and the otherheavy chain contains a variable region that binds to a second epitope,(ii) a combination of a pair of heavy chains which include only heavychains containing a variable region that binds to the first epitope, and(iii) a combination of a pair of heavy chains which include only heavychains containing a variable region that binds to the second epitope,which are present at a molecular ratio of 2:1:1. It is difficult topurify antigen-binding molecules containing the desired combination ofheavy chains from the mixture of three types of heavy chaincombinations.

When producing bispecific antibodies using such recombinationtechniques, bispecific antibodies containing a heteromeric combinationof heavy chains can be preferentially secreted by adding appropriateamino acid substitutions in the CH3 domains constituting the heavychains. Specifically, this method is conducted by substituting an aminoacid having a larger side chain (knob (which means “bulge”)) for anamino acid in the CH3 domain of one of the heavy chains, andsubstituting an amino acid having a smaller side chain (hole (whichmeans “void”)) for an amino acid in the CH3 domain of the other heavychain so that the knob is placed in the hole. This promotes heteromericheavy chain formation and simultaneously inhibits homomeric heavy chainformation (International Publication No. WO 1996027011; Ridgway et al.,Protein Engineering (1996) 9, 617-621; Merchant et al., NatureBiotechnology (1998) 16, 677-681).

Furthermore, there are also known techniques for producing a bispecificantibody by applying methods for controlling polypeptide association, orassociation of polypeptide-formed heteromeric multimers to theassociation between heavy chains. Specifically, methods for controllingheavy chain formation may be employed to produce a bispecific antibody(International Publication No. WO 2006/106905), in which amino acidresidues forming the interface between the heavy chains are altered toinhibit the association between the heavy chains having the samesequence and to allow the formation of heavy chains of differentsequences. Such methods can be used for generating bispecificantibodies.

In a non-limiting embodiment of the present invention, two polypeptidesconstituting an Fc region derived from a bispecific antibody describedabove can be suitably used as an Fc region to be included in theantigen-binding molecule. More specifically, it is preferable to use twopolypeptides that constitute an Fc region, and which comprise Cys forthe amino acid at position 349 and Trp for the amino acid at position366 according to EU numbering in the amino acid sequence of one of thepolypeptides; and Cys for the amino acid at position 356, Ser for theamino acid at position 366, Ala for the amino acid at position 368, andVal for the amino acid at position 407 as indicated by EU numbering inthe amino acid sequence of the other polypeptide.

In another non-limiting embodiment of the present invention, twopolypeptides that constitute an Fc region and which comprise Asp for theamino acid at position 409 according to EU numbering in the amino acidsequence of one of the polypeptides, and Lys for the amino acid atposition 399 according to EU numbering in the amino acid sequence of theother polypeptide, may be suitably used as the Fc region. In the aboveembodiment, the amino acid at position 409 may be Glu instead of Asp,and the amino acid at position 399 may be Arg instead of Lys. Moreover,in addition to the amino acid Lys at position 399, Asp may be suitablybe added as the amino acid at position 360 or Asp may suitably be addedas the amino acid at position 392.

In still another non-limiting embodiment of the present invention, twopolypeptides that constitute an Fc region, and which comprise Glu forthe amino acid at position 370 according to EU numbering in the aminoacid sequence of one of the polypeptides, and Lys for the amino acid atposition 357 according to EU numbering in the amino acid sequence of theother polypeptide, may be suitably used as the Fc region.

In yet another non-limiting embodiment of the present invention, twopolypeptides that constitute an Fc region, and which comprise Glu forthe amino acid at position 439 according to EU numbering in the aminoacid sequence of one of the polypeptides, and Lys for the amino acid atposition 356 according to EU numbering in the amino acid sequence of theother polypeptide, may be suitably used as the Fc region.

In still yet another non-limiting embodiment of the present invention,any of the embodiments indicated below of combinations from the abovemay be suitably used as the Fc region:

(i) two polypeptides that constitute an Fc region, and which compriseAsp for the amino acid at position 409 and Glu for the amino acid atposition 370 according to EU numbering in the amino acid sequence of oneof the polypeptides, and Lys for the amino acid at position 399 and Lysfor the amino acid at position 357 according to EU numbering in theamino acid sequence of the other polypeptide (in this embodiment, theamino acid at position 370 according to EU numbering may be Asp insteadof Glu, and the amino acid Asp at position 392 may be used instead ofthe amino acid Glu at position 370 according to EU numbering);

(ii) two polypeptides that constitute an Fc region, and which compriseAsp for the amino acid at position 409 and Glu for the amino acid atposition 439 according to EU numbering of the amino acid sequence of oneof the polypeptides; and Lys for the amino acid at position 399 and Lysfor the amino acid at position 356 according to EU numbering in theamino acid sequence of the other polypeptide (in this embodiment, theamino acid Asp at position 360, the amino acid Asp at position 392, orthe amino acid Asp at position 439 may be used instead of the amino acidGlu at position 439 according to EU numbering);

(iii) two polypeptides that constitute an Fc region, and which compriseGlu for the amino acid at position 370 and Glu for the amino acid atposition 439 according to EU numbering in the amino acid sequence of oneof the polypeptides, and Lys for the amino acid at position 357 and Lysfor the amino acid at position 356 according to EU numbering in theamino acid sequence of the other polypeptide; or

two polypeptides that constitute an Fc region, and which comprise Aspthe amino acid at position 409, Glu for the amino acid at position 370,and Glu for the amino acid at position 439 according to EU numbering inthe amino acid sequence of one of the polypeptides; and Lys for theamino acid at position 399, Lys for the amino acid at position 357, andLys for the amino acid at position 356 according to EU numbering in theamino acid sequence of the other polypeptide (in this embodiment, theamino acid at position 370 may not be substituted with Glu, andfurthermore, when the amino acid at position 370 is not substituted withGlu, the amino acid at position 439 may be Asp instead of Glu, or theamino acid Asp at position 392 may be used instead of the amino acid Gluat position 439, according to EU numbering).

Further, in another non-limiting embodiment of the present invention, itmay also be suitable to use two polypeptides that constitute an Fcregion, and which comprise Lys for the amino acid at position 356according to EU numbering in the amino acid sequence of one of thepolypeptides, and Arg for the amino acid at position 435 and Glu for theamino acid at position 439 according to EU numbering in the amino acidsequence of the other polypeptide.

In still another non-limiting embodiment of the present invention, itmay also be suitable to use two polypeptides that constitute an Fcregion and which comprise Lys for the amino acid at position 356 and Lysfor the amino acid at position 357 according to EU numbering in theamino acid sequence of one of the polypeptides, and Glu for the aminoacid at position 370, Arg for the amino acid at position 435, and Glufor the amino acid at position 439 according to EU numbering in theamino acid sequence of the other polypeptide.

Furthermore, in addition to the above-mentioned technologies ofassociating heterologous heavy chains, CrossMab technology which isknown as a technology for associating heterologous light chains, inwhich a light chain forming a variable region that binds to a firstepitope and a light chain forming a variable region that binds to asecond epitope are respectively associated with a heavy chain forming avariable region that binds to the first epitope and a heavy chainforming a variable region that binds to the second epitope (Scaefer etal. (Proc. Natl. Acad. Sci. U.S.A. (2011) 108, 11187-11192)), may alsobe used to produce the multispecific or multiparatopic antigen-bindingmolecules provided by the present invention. Furthermore, Fab-ArmExchange which is known as a technology for associating heterologousheavy chains, in which a heavy chain forming a variable region thatbinds to a first epitope and a heavy chain forming a variable regionthat binds to a second epitope by utilizing that heterologous IgG4 heavychains exchange each other (Labrijn et al. (Proc. Natl. Acad. Sci.U.S.A. (2013) 110, 5145-5150), WO2008119353), may also be used toproduce the multispecific or multiparatopic antigen-binding moleculesprovided by the present invention.

Effector Cells

In the present invention, the term “effector cells” may be used in thebroadest sense including T cells (CD4⁺ (helper lymphocyte) T cellsand/or CD8⁺ (cytotoxic) T cells), multinuclear leucocytes (neutrophils,eosinophils, basophils, mast cells), monocytes, macrophages,histiocytes, or leukocytes such as natural killer cells (NK cells),NK-like T cells, Kupffer cells, Langerhans cells, orlymphokine-activated killer cells (LAK cells), B-lymphocytes, orantigen-presenting cells such as dendritic cells or macrophages.Preferred examples of effector cells include CD8⁺ (cytotoxic) T cells,NK cells, or macrophages. Membrane-type molecules expressed on the cellmembrane of effector cells may be used as antigens to which at least oneantigen-binding domain contained in the antigen-binding molecule of thepresent invention binds. Non-limiting examples of a preferredmembrane-type molecule may be CD3, CD2, CD28, CD44, CD16, CD32, CD64, orNKG2D, NK cell-activating ligands, or polypeptides constituting TCR.

Cytotoxic Substances

In order for antigen-binding molecules of the present invention to bindto cancer cells and exhibit cytotoxic activity, cytotoxic substances maybe linked to antigen-binding molecules. The cytotoxic substances may bechemotherapeutic agents exemplified below, or compounds disclosed inCurr Opin Chem Biol (2010) 14, 529-37 and WO 2009/140242; and thesecompounds are linked to antigen-binding molecules by appropriate linkersand such. When antigen-binding molecules of the present invention areused as pharmaceutical compositions, these cytotoxic substances may belinked to the antigen-binding molecules prior to administration, or theymay be administered before, after, or at the same time when theantigen-binding molecules are administered to subjects (testindividuals, patients, and such).

The later-described modified antigen-binding molecules to whichcytotoxic substances such as chemotherapeutic agents, toxic peptides, orradioactive chemical substances have been linked may also be usedpreferably as antigen-binding molecules of the present invention havingcytotoxic activity. Such modified antigen-binding molecules (hereinafterreferred to as antigen-binding molecule-drug conjugate) can be obtainedby chemically modifying the obtained antigen-binding molecules. Methodsthat have been already established in the field of antibody-drugconjugates and such may be used appropriately as methods for modifyingantigen-binding molecules. Furthermore, a modified antigen-bindingmolecule to which a toxic peptide is linked can be obtained byexpressing in appropriate host cells a fused gene produced by linking agene encoding the toxic peptide in frame with a gene encoding anantigen-binding molecule of the present invention, and then isolating itfrom the cell culture.

Examples of chemotherapeutic agents linked to the antigen-bindingmolecules of the present invention may include:

azaribine, anastrozole, azacytidine, bleomycin, bortezomib,bryostatin-1, busulfan, camptothecin, 10-hydroxycamptothecin,carmustine, celebrex, chlorambucil, cisplatin, irinotecan, carboplatin,cladribine, cyclophosphamide, cytarabine, dacarbazine, docetaxel,dactinomycin, daunomycin glucuronide, daunorubicin, dexamethasone,diethylstilbestrol, doxorubicin, doxorubicin glucuronide, epirubicin,ethinyl estradiol, estramustine, etoposide, etoposide glucuronide,floxuridine, fludarabine, flutamide, fluorouracil, fluoxymesterone,gemcitabine, hydroxyprogesterone caproate, hydroxyurea, idarubicin,ifosfamide, leucovorin, lomustine, maytansinoid, mechlorethamine,medroxyprogesterone acetate, megestrol acetate, melphalan,mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin,mitotane, phenylbutyrate, prednisone, procarbazine, paclitaxel,pentostatin, semustine, streptozocin, tamoxifen, taxanes, taxol,testosterone propionate, thalidomide, thioguanine, thiotepa, teniposide,topotecan, uracil mustard, vinblastine, vinorelbine, and vincristine.

In the present invention, preferred chemotherapeutic agents arelow-molecular-weight chemotherapeutic agents. Low-molecular-weightchemotherapeutic agents are unlikely to interfere with the function ofantigen-binding molecules even after they bind to antigen-bindingmolecules of the present invention. In the present invention,low-molecular-weight chemotherapeutic agents usually have a molecularweight of 100 to 2000, preferably 200 to 1000. The chemotherapeuticagents exemplified herein are all low-molecular-weight chemotherapeuticagents. The chemotherapeutic agents of the present invention includeprodrugs that are converted into active chemotherapeutic agents in vivo.Prodrug activation may be enzymatic conversion or non-enzymaticconversion.

Moreover, cytotoxic substances that are linked to antigen-bindingmolecules of the present invention include, for example, toxic peptides(toxins) such as Pseudomonas exotoxin A, Saporin-s6, Diphtheria toxin,Cnidarian toxin; radioiodine; and photosensitizers. Suitable examples ofthe toxic peptides include the following:

Diphtheria toxin A Chain (Langone et al. (Methods in Enzymology (1983)93, 307-308)); Pseudomonas Exotoxin (Nature Medicine (1996) 2, 350-353);Ricin Chain (Ricin A Chain) (Fulton et al. (J. Biol. Chem. (1986) 261,5314-5319), Sivam et al. (Cancer Res. (1987) 47, 3169-3173), Cumber etal. (J. Immunol. Methods (1990) 135, 15-24), Wawrzynczak et al. (CancerRes. (1990) 50, 7519-7562), and Gheeite et al. (J. Immunol. Methods(1991) 142, 223-230));

Deglicosylated Ricin A Chain (Thorpe et al. (Cancer Res. (1987) 47,5924-5931));

Abrin A Chain (Wawrzynczak et al. (Br. J. Cancer (1992) 66, 361-366),Wawrzynczak et al. (Cancer Res. (1990) 50, 7519-7562), Sivam et al.(Cancer Res. (1987) 47, 3169-3173), and Thorpe et al. (Cancer Res.(1987) 47, 5924-5931));Gelonin (Sivam et al. (Cancer Res. (1987) 47, 3169-3173), Cumber et al.(J. Immunol. Methods (1990) 135, 15-24), Wawrzynczak et al. (CancerRes., (1990) 50, 7519-7562), and Bolognesi et al. (Clin. exp. Immunol.(1992) 89, 341-346));PAP-s; Pokeweed anti-viral protein from seeds (Bolognesi et al. (Clin.exp. Immunol. (1992) 89, 341-346));Briodin (Bolognesi et al. (Clin. exp. Immunol. (1992) 89, 341-346));Saporin (Bolognesi et al. (Clin. exp. Immunol. (1992) 89, 341-346));Momordin (Cumber et al. (J. Immunol. Methods (1990) 135, 15-24);Wawrzynczak et al. (Cancer Res. (1990) 50, 7519-7562); and Bolognesi etal. (Clin. exp. Immunol. (1992) 89, 341-346));Momorcochin (Bolognesi et al. (Clin. exp. Immunol. (1992) 89, 341-346));Dianthin 32 (Bolognesi et al. (Clin. exp. Immunol. (1992) 89, 341-346));Dianthin 30 (Stirpe F., Barbieri L. (FEBS letter (1986) 195, 1-8));Modeccin (Stirpe F., Barbieri L. (FEBS letter (1986) 195, 1-8));Viscumin (Stirpe F., Barbieri L. (FEBS letter (1986) 195, 1-8));Volkesin (Stirpe F., Barbieri L. (FEBS letter (1986) 195, 1-8));Dodecandrin (Stirpe F., Barbieri L. (FEBS letter (1986) 195, 1-8));Tritin (Stirpe F., Barbieri L. (FEBS letter (1986) 195, 1-8));Luffin (Stirpe F., Barbieri L. (FEBS letter (1986) 195, 1-8)); andTrichokirin (Casellas et al. (Eur. J. Biochem. (1988) 176, 581-588), andBolognesi et al. (Clin. exp. Immunol., (1992) 89, 341-346)).

Antigen-Binding Molecule

In the present invention, “an antigen-binding molecule comprising anantigen-binding domain whose antigen-binding activity in the presence ofa small molecule compound (e.g., target tissue-specific compound) ishigher than in the absence of the target tissue-specific compound” isused in the broadest sense; and specifically, it includes various typesof molecules as long as they show antigen-binding activity. Molecules inwhich an antigen-binding domain is linked to an Fc region include, forexample, antibodies. Antibodies may include single monoclonal antibodies(including agonistic antibodies and antagonistic antibodies), humanantibodies, humanized antibodies, chimeric antibodies, and such.Alternatively, when used as antibody fragments, they preferably includeantigen-binding domains and antigen-binding fragments (for example, Fab,F(ab′)2, scFv, and Fv). Scaffold molecules where three dimensionalstructures, such as already-known stable α/β barrel protein structure,are used as a scaffold (base) and only some portions of the structuresare made into libraries to construct antigen-binding domains are alsoincluded in antigen-binding molecules of the present invention.

An antigen-binding molecule of the present invention may contain atleast some portions of an Fc region that mediates the binding to Fcγreceptor and/or FcRn. In a non-limiting embodiment, the antigen-bindingmolecule includes, for example, antibodies and Fc fusion proteins. Afusion protein refers to a chimeric polypeptide comprising a polypeptidehaving a first amino acid sequence that is linked to a polypeptidehaving a second amino acid sequence that would not naturally link innature. For example, a fusion protein may comprise a polypeptidecomprising the amino acid sequence of at least a portion of an Fc region(for example, a portion of an Fc region responsible for the binding toFcγ receptor, and/or a portion of an Fc region responsible for thebinding to FcRn). The amino acid sequences may be present in separateproteins that are transported together to a fusion protein, or generallymay be present in a single protein; however, they are included in a newrearrangement in a fusion polypeptide. Fusion proteins can be produced,for example, by chemical synthesis, or by genetic recombinationtechniques to express a polynucleotide encoding peptide regions in adesired arrangement.

Respective domains of the present invention can be linked together vialinkers or directly via polypeptide binding. The linkers comprisearbitrary peptide linkers that can be introduced by genetic engineering,synthetic linkers, and linkers disclosed in, for example, Holliger etal., Protein Engineering (1996) 9(3), 299-305. However, peptide linkersare preferred in the present invention. The length of the peptidelinkers is not particularly limited, and can be suitably selected bythose skilled in the art according to the purpose. The length ispreferably five amino acids or more (without particular limitation, theupper limit is generally 30 amino acids or less, preferably 20 aminoacids or less), and particularly preferably 15 amino acids.

For example, such peptide linkers preferably include:

Ser Gly.Ser Gly.Gly.Ser Ser.Gly.Gly Gly.Gly.Gly.Ser (SEQ ID NO: 19)Ser.Gly.Gly.Gly (SEQ ID NO: 20) Gly.Gly.Gly.Gly.Ser (SEQ ID NO: 21)Ser.Gly.Gly.Gly.Gly (SEQ ID NO: 22) Gly.Gly.Gly.Gly.Gly.Ser (SEQ ID NO:23) Ser.Gly.Gly.Gly.Gly.Gly (SEQ ID NO: 24) Gly.Gly.Gly.Gly.Gly.Gly.Ser(SEQ ID NO: 25) Ser.Gly.Gly.Gly.Gly.Gly.Gly (SEQ ID NO: 26)(Gly.Gly.Gly.Gly.Ser (SEQ ID NO: 21))n (Ser.Gly.Gly.Gly.Gly (SEQ ID NO:22))n

where n is an integer of 1 or larger. The length or sequences of peptidelinkers can be selected accordingly by those skilled in the artdepending on the purpose.

Synthetic linkers (chemical crosslinking agents) is routinely used tocrosslink peptides, and for example:

N-hydroxy succinimide (NHS),disuccinimidyl suberate (DSS),bis(sulfosuccinimidyl) suberate (BS³),dithiobis(succinimidyl propionate) (DSP),dithiobis(sulfosuccinimidyl propionate) (DTSSP),ethylene glycol bis(succinimidyl succinate) (EGS),ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS),disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST),bis[2-(succinimidoxycarbonyloxy)ethyl] sulfone (BSOCOES),and bis[2-(sulfosuccinimidoxycarbonyloxy)ethyl] sulfone (sulfo-BSOCOES).These crosslinking agents are commercially available.

When multiple linkers for linking the respective domains are used, theymay all be of the same type, or may be of different types. In additionto the linkers exemplified above, linkers with peptide tags such as Histag, HA tag, myc tag, and FLAG tag may also be suitably used.Furthermore, hydrogen bonding, disulfide bonding, covalent bonding,ionic interaction, and properties of binding with each other as a resultof combination thereof may be suitably used. For example, the affinitybetween CH1 and CL of antibody may be used, and Fc regions originatingfrom the above-described bispecific antibodies may also be used forhetero Fc region association. Moreover, disulfide bonds formed betweendomains may also be suitably used.

In order to link respective domains via peptide linkage, polynucleotidesencoding the domains are linked together in frame. Known methods forlinking polynucleotides in frame include techniques such as ligation ofrestriction fragments, fusion PCR, and overlapping PCR. Such methods canbe appropriately used alone or in combination to constructantigen-binding molecules of the present invention. In the presentinvention, the terms “linked” and “fused”, or “linkage” and “fusion” areused interchangeably. These terms mean that two or more elements orcomponents such as polypeptides are linked together to form a singlestructure by any means including the above-described chemical linkingmeans and genetic recombination techniques. Fusing in frame means, whentwo or more elements or components are polypeptides, linking two or moreunits of reading frames to form a continuous longer reading frame whilemaintaining the correct reading frames of the polypeptides. When twomolecules of Fab are used as an antigen-binding domain, an antibody,which is an antigen-binding molecule of the present invention where theantigen-binding domain is linked in frame to a constant region includingan Fc region via peptide bond without linker, can be used as a preferredantigen-binding molecule of the present invention.

Low-Molecular-Weight Antibody

The antibodies used in the present invention are not limited tofull-length antibody molecules, and can be low-molecular-weightantibodies (minibodies) and modified products thereof. Alow-molecular-weight antibody includes an antibody fragment that lacks aportion of a full-length antibody (for example, whole antibody such aswhole IgG); and is not particularly limited as long as it has anantigen-binding activity. The low-molecular-weight antibody of thepresent invention is not particularly limited as long as it is a portionof a full-length antibody, but preferably comprises a heavy-chainvariable region (VH) and/or a light-chain variable region (VL). Theamino acid sequence of VH or VL may have substitution(s), deletion(s),addition(s), and/or insertion(s). Furthermore, as long as it has anantigen-binding activity, VH and/or VL can be partially deleted. Thevariable region may be chimerized or humanized. Specific examples ofantibody fragments include Fab, Fab′, F(ab′)2, and Fv. Specific examplesof low-molecular-weight antibodies include Fab, Fab′, F(ab′)2, Fv, scFv(single chain Fv), diabody, and sc(Fv)2 (single chain (Fv)2). Multimersof these antibodies (for example, dimers, trimers, tetramers, andpolymers) are also included in the low-molecular-weight antibodies ofthe present invention.

Antibody fragments can be produced by treating an antibody with anenzyme such as papain and pepsin. Alternatively, genes encoding theseantibody fragments can be constructed, inserted into expression vectors,and then expressed in appropriate host cells (see, for example, Co etal., (J. Immunol. (1994) 152, 2968-2976); Better and Horwitz (Methods inEnzymology (1989) 178, 476-496), Plueckthun and Skerra (Methods inEnzymology (1989) 178, 476-496); Lamoyi (Methods in Enzymology (1989)121, 652-663); Rousseaux (Methods in Enzymology (1989) 121, 663-669);and Bird, et al., TIBTECH (1991) 9, 132-137).

A diabody refers to a bivalent low-molecular-weight antibody constructedby gene fusion (Hollinger et al., (Proc. Natl. Acad. Sci. USA 90,6444-6448 (1993)); EP 404,097; WO 1993/11161; and such). A diabody is adimer composed of two polypeptide chains. Generally, in each polypeptidechain constituting the dimer, VL and VH are linked by a linker withinthe same chain. The linker in a diabody is generally short enough toprevent binding between VL and VH. Specifically, the amino acid residuesconstituting the linker are, for example, about five residues. A linkerbetween VL and VH that are encoded by the same polypeptide chain is tooshort to form a single-chain variable region fragment, and a dimer isformed between the polypeptide chains. As a result, diabodies have twoantigen binding sites.

scFv can be obtained by linking the H-chain V region and L-chain Vregion of an antibody. In scFv, the H-chain V region and L-chain Vregion are ligated via a linker, preferably a peptide linker (Huston, etal., Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 5879-5883). The H-chain Vregion and L-chain V region of scFv may be derived from any of theantibodies described herein. The peptide linker for ligating the Vregions is not particularly limited; and for example, any single-chainpeptide consisting of 3 to 25 residues or so, or peptide linkersdescribed later or such can be used as the linker. PCR methods such asthose described above can be used for ligating the V regions. DNAencoding scFv can be amplified by a PCR method using as a templateeither whole DNA or a partial DNA encoding a desired amino acidsequence, which is selected from a DNA sequence encoding the H chain orthe H chain V region of the above-mentioned antibody, and a DNA encodingthe L chain or the L chain V region of the above-mentioned antibody; andusing a pair of primers having sequences corresponding to the sequencesof the two ends. Next, a DNA having the desired sequence can be obtainedby performing a PCR reaction using a combination of a DNA encoding thepeptide linker portion, and a pair of primers having sequences designedso that both ends of the DNA will be ligated to the H chain and the Lchain, respectively. Once the scFv-encoding DNA is constructed,expression vectors having the DNA, and recombinant cells transformedwith the expression vector can be obtained according to conventionalmethods. Furthermore, the scFvs can be obtained by culturing theresulting recombinant cells to express the scFv-encoding DNA.

sc(Fv)2 is a low-molecular-weight antibody prepared by linking two VHsand two VLs with linkers or such to form a single chain (Hudson et al.(J. Immunol. Methods 1999; 231: 177-189)). sc(Fv)2 can be produced, forexample, by linking scFvs with a linker.

Moreover, antibodies in which two VHs and two VLs are arranged in theorder of VH, VL, VH, and VL starting from the N-terminal side of asingle chain polypeptide ([VH]-linker-[VL]-linker-[VH]-linker-[VL]) arepreferred. The order of the two VHs and the two VLs is not particularlylimited to the above-mentioned arrangement, and they may be arranged inany order. Examples include the following arrangements:

[VL]-linker-[VH]-linker-[VH]-linker-[VL][VH]-linker-[VL]-linker-[VL]-linker-[VH][VH]-linker-[VH]-linker-[VL]-linker-[VL][VL]-linker-[VL]-linker-[VH]-linker-[VH][VL]-linker-[VH]-linker-[VL]-linker-[VH]

A linker similar to the linker described in the section “Antigen-bindingmolecules” above may be used as the linker for linking the antibodyvariable regions. A particularly preferred embodiment of sc(Fv)2 in thepresent invention includes, for example, the following sc(Fv)2:

[VH]-peptide linker (15 amino acids)-[VL]-peptide linker (15 aminoacids)-[VH]-peptide linker (15 amino acids)-[VL]

Typically, three linkers are required to link four antibody variableregions. The linkers to be used may be of the same type or differenttypes. Examples of a non-limiting embodiment of a low-molecular-weightantibody in the present invention include a diabody or sc(Fv)2, whereinthe paratopes are different from each other; one of the paratopes bindsto an epitope in a membrane-type molecule which binds to a cell membraneof cancer cells, cells infiltrated into inflammatory tissues, and such;and the other paratope binds to an epitope in the membrane-type moleculeexpressed on the cell membrane of effector cells. In the above-mentioneddiabody or sc(Fv)2, the binding activity of one of the paratopes towardan epitope in a membrane-type molecule which binds to a cell membrane ofcancer cells, cells infiltrated into inflammatory tissues, and such maydepend on a small molecule compound (e.g., cancer tissue-specificcompound, inflammatory tissue-specific compound, or unnatural compound),the binding activity of one of the paratopes toward an epitope in amembrane-type molecule which binds to an effector cell membrane maydepend on a small molecule compound (e.g., a cancer tissue-specificcompound, inflammatory tissue-specific compound, or unnatural compound),or the binding activities of both paratopes may depend on a smallmolecule compound (e.g., a cancer tissue-specific compound, inflammatorytissue-specific compound, or unnatural compound).

A non-limiting embodiment of a low-molecular-weight antibody in thepresent invention includes, for example, a diabody or sc(Fv)2, whereinthe paratopes are different from each other; one of the paratopes bindsto an epitope in a membrane-type molecule which binds to a cell membraneof cancel cells, cells infiltrated into inflammatory tissues, and such;and the other paratope binds to an epitope in a cytotoxic substance. Inthe diabody or sc(Fv)2 mentioned above, the binding activity of one ofthe paratopes that binds to an epitope in a membrane-type molecule whichbinds to a cell membrane of cancel cells, cells infiltrated intoinflammatory tissues, and such may depend on a small molecule compound(e.g., a cancer tissue-specific compound, inflammatory tissue-specificcompound, or unnatural compound), the binding activity of the otherparatope that binds to an epitope in a cytotoxic substance may depend ona small molecule compound (e.g., a cancer tissue-specific compound,inflammatory tissue-specific compound, or unnatural compound), or thebinding activities of both paratopes may depend on a cancertissue-specific compound.

Such low-molecular-weight antibody can be obtained by treating anantibody with an enzyme such as papain or pepsin to generate antibodyfragments, or by constructing DNAs that encode these antibody fragmentsor low-molecular-weight antibodies, inserting them into expressionvectors, and then expressing them in appropriate host cells (see, forexample, Co, M. S. et al., J. Immunol. (1994) 152, 2968-2976; Better, M.and Horwitz, A. H., Methods Enzymol. (1989) 178, 476-496; Pluckthun, A.and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E.,Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al., MethodsEnzymol. (1986) 121, 663-669; and Bird, R. E. and Walker, B. W., TrendsBiotechnol. (1991) 9, 132-137).

FcRn

Unlike Fcγ receptor belonging to the immunoglobulin superfamily, humanFcRn is structurally similar to polypeptides of major histocompatibilitycomplex (MHC) class I, exhibiting 22% to 29% sequence identity to classI MHC molecules (Ghetie el al., Immunol. Today (1997) 18 (12): 592-598).FcRn is expressed as a heterodimer consisting of soluble p or lightchain (β2 microglobulin) complexed with transmembrane α or heavy chain.Like MHC, FcRn α chain comprises three extracellular domains (α1, α2,and α3) and its short cytoplasmic domain anchors the protein onto thecell surface. α1 and α2 domains interact with the FcRn-binding domain ofthe antibody Fc region (Raghavan et al., Immunity (1994) 1: 303-315).

FcRn is expressed in maternal placenta and yolk sac of mammals, and isinvolved in mother-to-fetus IgG transfer. In addition, in neonatal smallintestine of rodents, where FcRn is expressed, FcRn is involved intransfer of maternal IgG across brush border epithelium from ingestedcolostrum or milk. FcRn is expressed in a variety of other tissues andendothelial cell systems of various species. FcRn is also expressed inadult human endothelia, muscular blood vessels, and hepatic sinusoidalcapillaries. FcRn is believed to play a role in maintaining the plasmaIgG concentration by mediating recycling of IgG to serum upon binding toIgG. Typically, binding of FcRn to IgG molecules is strictly pHdependent. The optimal binding is observed in an acidic pH range below7.0.

Human FcRn whose precursor is a polypeptide having the signal sequenceof SEQ ID NO: 28 (the polypeptide with the signal sequence is shown inSEQ ID NO: 29) forms a complex with human β2-microglobulin in vivo.Soluble human FcRn complexed with β2-microglobulin is produced by usingconventional recombinant expression techniques. Fc regions of thepresent invention can be assessed for their binding activity to such asoluble human FcRn complexed with β2-microglobulin. Herein, unlessotherwise specified, human FcRn refers to a form capable of binding toan Fc region of the present invention. Examples include a complexbetween human FcRn and human β2-microglobulin.

Embodiments of combining the present invention with techniques formodifying the constant region are, for example, combinations withantibody modification techniques such as Fc-modifying techniques toenhance FcRn binding at acidic pH (WO2002060919, WO2004035752, andWO2000042072), Fc-modifying techniques to enhance FcRn binding atneutral pH (WO2011122011 and WO2012133782), techniques for enhancinginhibitory Fcγ receptor-selective binding (WO2012115241 andWO2013125667), techniques for enhancing activating Fcγreceptor-selective binding (techniques for enhancing ADCC activity)(WO2013002362), and techniques for lowering the binding activity to aRheumatoid factor (WO2013046704).

A non-limiting embodiment of a combination of the present invention withtechniques for modifying the variable region includes, for example,combinations with techniques for modifying pH-dependent antibodies(WO2009125825), calcium-dependent antibodies (WO2012073992), and such.

Heterocomplex Comprising the Four Molecules Including Two Molecules ofFcRn and One Molecule of Activating Fcγ Receptor

Crystallographic studies on FcRn with IgG antibodies demonstrated thatan FcRn-IgG complex is composed of one molecule of IgG for two moleculesof FcRn, and the two molecules are thought to bind around the interfaceof the CH2 and CH3 domains located on both sides of the IgG Fc region(Burmeister et al. (Nature (1994) 372, 336-343)). Meanwhile, asdemonstrated in Example 3 of PCT/JP2012/058603, the antibody Fc regionwas demonstrated to be able to form a complex comprising the fourmolecules including two molecules of FcRn and one molecule of activatingFcγ receptor (PCT/JP2012/058603). This heterocomplex formation is aphenomenon which was revealed as a result of analyzing the properties ofantigen-binding molecules containing an Fc region having an FcRn-bindingactivity under a neutral pH range condition.

While the present invention is not bound to a particular principle, itcan be considered that antigen-binding molecules administered in vivoproduce the effects described below on the in vivo pharmacokinetics(plasma retention) of the antigen-binding molecules and an immuneresponse (immunogenicity) to the administered antigen-binding molecules,as a result of the formation of heterocomplexes containing the fourmolecules including the Fc region contained in the antigen-bindingmolecules, two molecules of FcRn, and one molecule of activating Fcγreceptor. In addition to the various types of activating Fcγ receptors,FcRn is expressed on immune cells. It is suggested that the formation ofsuch tetrameric complexes on immune cells by antigen-binding moleculespromotes incorporation of antigen-binding molecules into immune cells byincreasing affinity toward immune cells and by causing association ofintracellular domains to enhance the internalization signal. The samealso applies to antigen-presenting cells and the possibility thatantigen binding-molecules are likely to be incorporated intoantigen-presenting cells by formation of tetrameric complexes on thecell membrane of antigen-presenting cells. In general, antigen-bindingmolecules incorporated into antigen-presenting cells are degraded in thelysosomes of the antigen-presenting cells and are presented to T cells.As a result, plasma retention of antigen-binding molecules may beworsened because incorporation of antigen-binding molecules intoantigen-presenting cells is promoted by the formation of theabove-described tetrameric complexes on the cell membrane of theantigen-presenting cells. Similarly, an immune response may be induced(aggravated).

For this reason, it is conceivable that when an antigen-binding moleculehaving lowered ability to form such tetrameric complexes is administeredin vivo, plasma retention of the antigen-binding molecules wouldimprove, and induction of in vivo immune response would be suppressed.Preferred embodiments of such antigen-binding molecules which inhibitthe formation of these complexes on immune cells includingantigen-presenting cells are, for example, the three embodimentsdescribed below.

Antigen-Binding Molecules which Inhibit the Formation of HeterocomplexesEmbodiment 1 An Antigen-Binding Molecule Containing an Fc Region HavingFcRn-Binding Activity Under a Neutral pH Range Condition and WhoseBinding Activity Toward Activating FcγR is Lower than the BindingActivity of a Native Fc Region Toward Activating FcγR

The antigen-binding molecule of Embodiment 1 forms a trimeric complex bybinding to two molecules of FcRn; however, it does not form any complexcontaining activating FcγR. An Fc region whose binding activity towardactivating FcγR is lower than the binding activity of a native Fc regiontoward activating FcγR can be prepared by altering the amino acids ofthe native Fc region as described above. Whether the binding activitytoward activating FcγR of the altered Fc region is lower than thebinding activity toward activating FcγR of the native Fc region can beappropriately tested using the methods described in the section “BindingActivity” above.

Preferred activating Fcγ receptors include FcγRI (CD64) which includesFcγRIa, FcγRIb, and FcγRIc; FcγRIIa (including allotypes R131 and H131);and FcγRIII (CD16) which includes isoforms FcγRIIIa (including allotypesV158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 andFcγRIIIb-NA2).

Herein, “a binding activity of the Fc region variant toward anactivating Fcγ receptor is lower than the binding activity of the nativeFc region toward an activating Fcγ receptor” means that the bindingactivity of the Fc region variant toward any of the human Fcγ receptors(FcγRI, FcγRIIa, FcγRIIIa, and/or FcγRIIIb) is lower than the bindingactivity of the native Fc region toward these human Fcγ receptors. Forexample, it means that based on an above-described analytical method,the binding activity of the antigen-binding molecule containing an Fcregion variant as compared to the binding activity of an antigen-bindingmolecule containing a native Fc region as a control is 95% or less,preferably 90% or less, 85% or less, 80% or less, 75% or less, andparticularly preferably 70% or less, 65% or less, 60% or less, 55% orless, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less,25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% orless, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% orless, or 1% or less. As a native Fc region, a starting Fc region may beused, and Fc regions of wild-type antibodies of different isotypes mayalso be used.

Meanwhile, the binding activity of the native form toward an activatingFcγR is preferably a binding activity toward the Fcγ receptor for humanIgG1. Other than performing the above-described alterations, bindingactivity toward the Fcγ receptor can be lowered by changing the isotypeto human IgG2, human IgG3, or human IgG4. Alternatively, besides byperforming the above-described alterations, the binding activity towardan Fcγ receptor can also be lowered by expressing the antigen-bindingmolecule containing an Fc region having a binding activity toward theFcγ receptor in hosts that do not add sugar chains such as Escherichiacoli.

For the antigen-binding molecule containing a control Fc region, anantigen-binding molecule having an Fc region of a monoclonal IgGantibody may be appropriately used. The structures of such Fc regionsare shown in SEQ ID NO: 5 (A is added to the N terminus of RefSeqAccession No. AAC82527.1), SEQ ID NO: 6 (A is added to the N terminus ofRefSeq Accession No. AAB59393.1), SEQ ID NO: 7 (RefSeq Accession No.CAA27268.1), and SEQ ID NO: 8 (A is added to the N terminus of RefSeqAccession No. AAB59394.1). Further, when an antigen-binding moleculecontaining an Fc region of a particular antibody isotype is used as thetest substance, effect on the binding activity of the antigen-bindingmolecule containing the Fc region toward an Fcγ receptor is tested byusing the antigen-binding molecule having an Fc region of a monoclonalIgG antibody of a particular isotype as a control. In this way,antigen-binding molecules containing an Fc region whose binding activitytoward the Fcγ receptor was demonstrated to be high are suitablyselected.

In a non-limiting embodiment of the present invention, preferredexamples of Fc regions whose binding activity toward an activating FcγRis lower than the binding activity of the native Fc region toward anactivating FcγR include Fc regions with alteration of one or more aminoacids at any of positions 234, 235, 236, 237, 238, 239, 270, 297, 298,325, 328, and 329 as indicated by EU numbering in the amino acids of anabove-described Fc region to be different from those of the native Fcregion. The alterations in the Fc region are not limited to the aboveexample, and they may be, for example, modifications such asdeglycosylation (N297A and N297Q), IgG1-L234A/L235A,IgG1-A325A/A330S/P331S, IgG1-C226S/C229S,IgG1-C226S/C229S/E233P/L234V/L235A, IgG1-L234F/L235E/P331S,IgG1-S267E/L328F, IgG2-V234A/G237A, IgG2-H268Q/V309L/A330S/A331S,IgG4-L235A/G237A/E318A, and IgG4-L236E described in Cur. Opin. inBiotech. (2009) 20 (6), 685-691; alterations such as G236R/L328R,L235G/G236R, N325A/L328R, and N325L/L328R described in WO 2008/092117;amino acid insertions at positions 233, 234, 235, and 237 according toEU numbering; and alterations at the positions described in WO2000/042072.

In a non-limiting embodiment of the present invention, examples of apreferred Fc region include Fc regions having one or more of thefollowing alterations as indicated by EU numbering in an aforementionedFc region:

Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Met, Phe, Pro, Ser, Thr, orTrp for the amino acid at position 234;Ala, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Pro, Ser, Thr, Val, orArg for the amino acid at position 235;Arg, Asn, Gln, His, Leu, Lys, Met, Phe, Pro, or Tyr for the amino acidat position 236;Ala, Asn, Asp, Gln, Glu, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Val,Tyr, or Arg for the amino acid at position 237;Ala, Asn, Gln, Glu, Gly, His, Ile, Lys, Thr, Trp, or Arg for the aminoacid at position 238;Gln, His, Lys, Phe, Pro, Trp, Tyr, or Arg for the amino acid at position239;Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp,Tyr, or Val for the amino acid at position 265;Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Phe, Pro, Ser, Thr, Trp, orTyr for the amino acid at position 266;Arg, His, Lys, Phe, Pro, Trp, or Tyr for the amino acid at position 267;Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val for the amino acid at position 269;Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val for the amino acid at position 270;Arg, His, Phe, Ser, Thr, Trp, or Tyr for the amino acid at position 271;Arg, Asn, Asp, Gly, His, Phe, Ser, Trp, or Tyr for the amino acid atposition 295;Arg, Gly, Lys, or Pro for the amino acid at position 296;Ala for the amino acid at position 297;Arg, Gly, Lys, Pro, Trp, or Tyr for the amino acid at position 298;Arg, Lys, or Pro for the amino acid at position 300;Lys or Pro for the amino acid at position 324;Ala, Arg, Gly, His, Ile, Lys, Phe, Pro, Thr, Trp, Tyr, or Val for theamino acid at position 325;Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Valfor the amino acid at position 327;Arg, Asn, Gly, His, Lys, or Pro for the amino acid at position 328;Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp,Tyr, Val, or Arg for the amino acid at position 329;Pro or Ser for the amino acid at position 330;Arg, Gly, or Lys for the amino acid at position 331; orArg, Lys, or Pro for the amino acid at position 332.

Embodiment 2 An Antigen-Binding Molecule Containing an Fc Region HavingFcRn-Binding Activity Under a Neutral pH Range Condition and WhoseBinding Activity Toward an Inhibitory FcγR is Higher than the BindingActivity Toward an Activating Fcγ Receptor

By binding to two molecules of FcRn and one molecule of inhibitory FcγR,the antigen-binding molecule of Embodiment 2 can form a complexcomprising these four molecules. However, since a single antigen-bindingmolecule can bind with only one molecule of FcγR, the singleantigen-binding molecule in a state bound to an inhibitory FcγR cannotbind to other activating FcγRs. Furthermore, it has been reported thatan antigen-binding molecule that is incorporated into cells in a statebound to an inhibitory FcγR is recycled onto the cell membrane, and thusescapes from degradation inside the cells (Immunity (2005) 23, 503-514).More specifically, it is considered that antigen-binding moleculeshaving selective binding activity toward an inhibitory FcγR cannot formheterocomplexes containing an activating FcγR and two molecules of FcRn,which cause an immune response.

Preferred activating Fcγ receptors include FcγRI (CD64) which includesFcγRIa, FcγRIb, and FcγRIc; FcγRIIa (including allotypes R131 and H131);and FcγRIII (CD16) which includes isoforms FcγRIIIa (including allotypesV158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 andFcγRIIIb-NA2). Meanwhile, examples of preferred inhibitory Fcγ receptorsinclude FcγRIIb (including FcγRIIb-1 and FcγRIIb-2).

Herein, “a binding activity toward an inhibitory FcγR is higher than thebinding activity toward an activating Fcγ receptor” means that thebinding activity of the Fc region variant toward FcγRIIb is higher thanthe binding activity toward any of the human Fcγ receptors, FcγRI,FcγRIIa, FcγRIIIa, and/or FcγRIIIb. For example, it means that based onan above-described analytical method, the binding activity towardFcγRIIb of the antigen-binding molecule containing an Fc region variantas compared with the binding activity toward any of the human Fcγreceptors, FcγRI, FcγRIIa, FcγRIIIa, and/or FcγRIIIb is 105% or more,preferably 110% or more, 120% or more, 130% or more, 140% or more, andparticularly preferably 150% or more, 160% or more, 170% or more, 180%or more, 190% or more, 200% or more, 250% or more, 300% or more, 350% ormore, 400% or more, 450% or more, 500% or more, 750% or more, 10 timesor more, 20 times or more, 30 times or more, 40 times or more, 50 timesor more.

Most preferably, the binding activity toward FcγRIIb is higher than eachof the binding activities toward FcγRIa, FcγRIIa (including allotypesR131 and H131), and FcγRIIIa (including allotypes V158 and F158). FcγRIashows a markedly high affinity toward native IgG1; thus, the binding isthought to be saturated in vivo due to the presence of a large amount ofendogenous IgG1. For this reason, inhibition of complex formation may bepossible even if the binding activity toward FcγRIIb is greater than thebinding activities toward FcγRIIa and FcγRIIIa, and lower than thebinding activity toward FcγRIa.

As a control antigen-binding molecule containing an Fc region,antigen-binding molecules having an Fc region of a monoclonal IgGantibody may be appropriately used. The structures of such Fc regionsare shown in SEQ ID NO: 5 (A is added to the N terminus of RefSeqAccession No. AAC82527.1), SEQ ID NO: 6 (A is added to the N terminus ofRefSeq Accession No. AAB59393.1), SEQ ID NO: 7 (RefSeq Accession No.CAA27268.1), and SEQ ID NO: 8 (A is added to the N terminus of RefSeqAccession No. AAB59394.1). Further, when an antigen-binding moleculecontaining an Fc region of a particular antibody isotype is used as thetest substance, effect on the binding activity of the Fcregion-containing antigen-binding molecule toward an Fcγ receptor istested by using an antigen-binding molecule having the Fc region of amonoclonal IgG antibody of a particular isotype as a control. In thisway, antigen-binding molecules containing an Fc region whose bindingactivity toward the Fcγ receptor was demonstrated to be high areappropriately selected.

In a non-limiting embodiment of the present invention, preferredexamples of Fc regions having a selective binding activity toward aninhibitory FcγR include Fc regions in which among the amino acids of anabove-described Fc region, the amino acid at 238 or 328 as indicated byEU numbering is altered to an amino acid different from that of thenative Fc region. Furthermore, as an Fc region having a selectivebinding activity toward an inhibitory Fcγ receptor, the Fc regions oralterations described in US 2009/0136485 can be appropriately selected.

In a non-limiting embodiment of the present invention, a preferredexample is an Fc region having one or more of the following alterationsas indicated by EU numbering in an aforementioned Fc region: the aminoacid at position 238 is Asp; or the amino acid at position 328 is Glu.

In still another non-limiting embodiment of the present invention,examples of a preferred Fc region include Fc regions having asubstitution of Pro at position 238 according to EU numbering with Aspand having one or more of the alterations:

alteration of the amino acid at position 237 according to EU numberingto Trp, the amino acid at position 237 according to EU numbering is Phe,the amino acid at position 267 according to EU numbering is Val, theamino acid at position 267 according to EU numbering is Gln, the aminoacid at position 268 according to EU numbering is Asn, the amino acid atposition 271 according to EU numbering is Gly, the amino acid atposition 326 according to EU numbering is Leu, the amino acid atposition 326 according to EU numbering is Gln, the amino acid atposition 326 according to EU numbering is Glu, the amino acid atposition 326 according to EU numbering is Met, the amino acid atposition 239 according to EU numbering is Asp, the amino acid atposition 267 according to EU numbering is Ala, the amino acid atposition 234 according to EU numbering is Trp, the amino acid atposition 234 according to EU numbering is Tyr, the amino acid atposition 237 according to EU numbering is Ala, the amino acid atposition 237 according to EU numbering is Asp, the amino acid atposition 237 according to EU numbering is Glu, the amino acid atposition 237 according to EU numbering is Leu, the amino acid atposition 237 according to EU numbering is Met, the amino acid atposition 237 according to EU numbering is Tyr, the amino acid atposition 330 according to EU numbering is Lys, the amino acid atposition 330 according to EU numbering is Arg, the amino acid atposition 233 according to EU numbering is Asp, the amino acid atposition 268 according to EU numbering is Asp, the amino acid atposition 268 according to EU numbering is Glu, the amino acid atposition 326 according to EU numbering is Asp, the amino acid atposition 326 according to EU numbering is Ser, the amino acid atposition 326 according to EU numbering is Thr, the amino acid atposition 323 according to EU numbering is Ile, the amino acid atposition 323 according to EU numbering is Leu, the amino acid atposition 323 according to EU numbering is Met, the amino acid atposition 296 according to EU numbering is Asp, the amino acid atposition 326 according to EU numbering is Ala, the amino acid atposition 326 according to EU numbering is Asn, and the amino acid atposition 330 according to EU numbering is Met.

Embodiment 3 An Antigen-Binding Molecule Containing an Fc Region, inwhich One of the Two Polypeptides Constituting the Fc Region has anFcRn-Binding Activity Under a Neutral pH Range Condition and the OtherPolypeptide does not have FcRn-Binding Activity Under a Neutral pH RangeCondition

By binding to one molecule of FcRn and one molecule of FcγR, theantigen-binding molecule of Embodiment 3 can form a trimeric complex;however, it does not form any heterocomplex comprising four moleculesincluding two molecules of FcRn and one molecule of FcγR. As an Fcregion in which one of the two polypeptides constituting the Fc regionhas an FcRn-binding activity under a neutral pH range condition and theother does not have any FcRn-binding activity under a neutral pH rangecondition contained in the antigen-binding molecule of Embodiment 3, Fcregions derived from bispecific antibodies may be suitably used.Bispecific antibodies are two types of antibodies having specificitiestoward different antigens. Bispecific antibodies of an IgG type can besecreted from hybrid hybridomas (quadromas) resulting from fusion of twotypes of hybridomas producing IgG antibodies (Milstein et al. (Nature(1983) 305, 537-540).

When an antigen-binding molecule of Embodiment 3 described above isproduced by using recombination techniques such as those described inthe section “Antibodies” above, one can use a method in which genesencoding the polypeptides that constitute the two types of Fc regions ofinterest are transfected into cells to co-express them. However, theproduced Fc regions will be a mixture in which the following will existat a molecular ratio of 2:1:1: an Fc region in which one of the twopolypeptides constituting the Fc region has an FcRn-binding activityunder a neutral pH range condition and the other polypeptide does nothave any FcRn-binding activity under a neutral pH range condition; an Fcregion in which the two polypeptides constituting the Fc region bothhave an FcRn-binding activity under a neutral pH range condition; and anFc region in which both of the two polypeptides constituting the Fcregion do not have FcRn-binding activity under a neutral pH rangecondition. It is difficult to purify antigen-binding moleculescontaining the desired combination of Fc regions from the three types ofIgGs.

When producing the antigen-binding molecules of Embodiment 3 using suchrecombination techniques, antigen-binding molecules comprising aheteromeric combination of Fc regions can be preferentially secreted byadding appropriate amino acid substitutions to the CH3 domainsconstituting the Fc regions. Specifically, this method is conducted bysubstituting an amino acid having a larger side chain (knob (which means“bulge”)) for an amino acid in the CH3 domain of one of the heavychains, and substituting an amino acid having a smaller side chain (hole(which means “void”)) for an amino acid in the CH3 domain of the otherheavy chain so that the knob is arranged in the hole. This promotesheteromeric H chain formation and simultaneously inhibits homomeric Hchain formation (WO 1996027011; Ridgway et al., (Protein Engineering(1996) 9, 617-621); Merchant et al., (Nature Biotechnology (1998) 16,677-681)).

Furthermore, there are also known techniques for producing a bispecificantibody by applying methods for controlling polypeptide association orassociation of polypeptide-formed heteromeric multimers to theassociation between two polypeptides that constitute an Fc region.Specifically, methods for controlling polypeptide association may beemployed to produce a bispecific antibody (WO 2006/106905), in whichamino acid residues forming the interface between two polypeptides thatconstitute the Fc region are altered to inhibit the association betweenFc regions having the same sequence, and to allow the formation ofpolypeptide complexes formed by two Fc regions of different sequences.Specifically, the methods in the above-described section on bispecificantibodies and methods for producing them can be used as a non-limitingembodiment for preparing the antigen-binding molecule of Embodiment 3 ofthe present invention.

These antigen-binding molecules of Embodiments 1 to 3 are all expectedto be able to reduce immunogenicity and improve plasma retention ascompared to antigen-binding molecules capable of forming tetramericcomplexes.

Methods for Producing Antigen-Binding Domains

The present invention provides methods for producing antigen-bindingdomains whose antigen-binding activity in the presence of a smallmolecule compound is higher than the antigen-binding activity in theabsence of the compound.

More specifically, the present invention provides a method for producingan antigen-binding domain, which comprises steps (a) to (e) below:

(a) determining the antigen-binding activity of an antigen-bindingdomain in the absence of a small molecule compound;(b) determining the antigen-binding activity of an antigen-bindingdomain in the presence of the small molecule compound;(c) selecting an antigen-binding domain whose antigen-binding activityin the absence of a small molecule compound is lower than in thepresence of the compound;(d) culturing cells transfected with a vector to which a polynucleotideencoding the antigen-binding domain selected in (c) is operably linked;and(e) collecting an antigen-binding domain from a culture medium of thecells cultured in (d).

The present invention also provides a method for producing anantigen-binding domain, which comprises steps (a) to (e) below:

(a) determining the antigen-binding activity of an antigen-bindingdomain in the presence of a low concentration of a small moleculecompound;(b) determining the antigen-binding activity of an antigen-bindingdomain in the presence of a high concentration of the small moleculecompound;(c) selecting an antigen-binding domain whose antigen-binding activityin the presence of a low concentration of the small molecule compound islower than in the presence of a high concentration of the compound;(d) culturing cells transfected with a vector to which a polynucleotideencoding the antigen-binding domain selected in (c) is operably linked;and(e) collecting an antigen-binding domain from a culture medium of thecells cultured in (d).

Furthermore, the present invention provides a method for producing anantigen-binding domain, which comprises steps (a) to (e) below:

(a) contacting antigen-binding domains or a library thereof with anantigen in the presence of a small molecule compound;(b) placing the antigen-binding domains that bound to the antigen insaid step (a) in the absence of the compound;(c) isolating an antigen-binding domain that was dissociated in saidstep (b);(d) culturing cells transfected with a vector to which a polynucleotideencoding the antigen-binding domain selected in (c) is operably linked;and(e) collecting an antigen-binding domain from a culture medium of thecells cultured in (d).

In addition, the present invention provides a method for producing anantigen-binding domain, which comprises steps (a) to (e) below:

(a) contacting antigen-binding domains or a library thereof to anantigen in the presence of a high concentration of a small moleculecompound;(b) placing the antigen-binding domains that bind to the antigen in saidstep (a) in the presence of a low concentration of the compound;(c) isolating an antigen-binding domain that dissociates in said step(b);(d) culturing cells transfected with a vector to which a polynucleotideencoding the antigen-binding domain selected in (c) is operably linked;and(e) collecting an antigen-binding domain from a culture medium of thecells cultured in (d).

The present invention provides a method for producing an antigen-bindingdomain, which comprises steps of (a) to (f) below:

(a) contacting a library of antigen-binding domains with an antigen inthe absence of a small molecule compound;(b) selecting antigen-binding domains that do not bind to the antigen insaid step (a);(c) allowing the antigen-binding domains selected in said step (b) tobind to the antigen in the presence of the compound;(d) isolating an antigen-binding domain that bind to the antigen in saidstep (c);(e) culturing cells transfected with a vector to which a polynucleotideencoding the antigen-binding domain selected in (d) is operably linked;and(f) collecting an antigen-binding domain from a culture medium of thecells cultured in (e).

The present invention provides a method for producing an antigen-bindingdomain, which comprises steps (a) to (f) below:

(a) contacting a library of antigen-binding domains with an antigen inthe presence of a low concentration of a small molecule compound;(b) selecting antigen-binding domains that do not bind to the antigen insaid step (a);(c) allowing the antigen-binding domains selected in said step (b) tobind to the antigen in the presence of a high concentration of thecompound;(d) isolating an antigen-binding domain that bind to the antigen in saidstep (c);(e) culturing cells transfected with a vector to which a polynucleotideencoding the antigen-binding domain selected in (d) is operably linked;and(f) collecting an antigen-binding domain from a culture medium of thecells cultured in (e).

The present invention provides a method for producing an antigen-bindingdomain, which comprises steps (a) to (e) below:

(a) contacting a library of antigen-binding domains with anantigen-immobilized column in the presence of a small molecule compound;(b) eluting antigen-binding domains that bind to the column in said step(a) from the column in the absence of the compound;(c) isolating the antigen-binding domain eluted in said step (b);(d) culturing cells transfected with a vector to which a polynucleotideencoding the antigen-binding domain selected in (c) is operably linked;and(e) collecting an antigen-binding domain from a culture medium of thecells cultured in (d).

The present invention provides a method for producing an antigen-bindingdomain, which comprises steps (a) to (e) below:

(a) contacting a library of antigen-binding domains with anantigen-immobilized column in the presence of a high concentration of asmall molecule compound;(b) eluting antigen-binding domains that bind to the column in said step(a) from the column in the presence of a low concentration of thecompound;(c) isolating an antigen-binding domain eluted in said step (b);(d) culturing cells transfected with a vector to which a polynucleotideencoding the antigen-binding domain selected in (c) is operably linked;and(e) collecting an antigen-binding domain from a culture medium of thecells cultured in (d).

The present invention provides a method for producing an antigen-bindingdomain, which comprises steps (a) to (f) below:

(a) allowing a library of antigen-binding domains to pass through anantigen-immobilized column in the absence of a small molecule compound;(b) collecting antigen-binding domains that are eluted without bindingto the column in step (a);(c) allowing the antigen-binding domains collected in step (b) to bindto the antigen in the presence of the compound;(d) isolating an antigen-binding domain that bind to the antigen in step(c);(e) culturing cells transfected with a vector to which a polynucleotideencoding the antigen-binding domain selected in (d) is operably linked;and(f) collecting an antigen-binding domain from a culture medium of thecells cultured in (e).

The present invention provides a method for producing an antigen-bindingdomain, which comprises steps (a) to (f) below:

(a) allowing a library of antigen-binding domains to pass through anantigen-immobilized column in the presence of a low concentration of asmall molecule compound;(b) collecting antigen-binding domains that are eluted without bindingto the column in said step (a);(c) allowing the antigen-binding domains collected in said step (b) tobind to the antigen in the presence of a high concentration of thecompound;(d) isolating an antigen-binding domain that binds to the antigen insaid step (c);(e) culturing cells transfected with a vector to which a polynucleotideencoding the antigen-binding domain selected in (d) is operably linked;and(f) collecting an antigen-binding domain from a culture medium of thecells cultured in (e).

Furthermore, the present invention provides a method for producing anantigen-binding domain, which comprises steps (a) to (f) below:

(a) contacting an antigen with a library of antigen-binding domains inthe presence of a small molecule compound;(b) obtaining antigen-binding domains that bind to the antigen in step(a);(c) placing the antigen-binding domain obtained in step (b) in theabsence of the compound;(d) isolating an antigen-binding domain whose antigen-binding activityin step (c) is weaker than the reference selected in step (b);(e) culturing cells transfected with a vector to which a polynucleotideencoding the antigen-binding domain selected in (d) is operably linked;and(f) collecting an antigen-binding domain from a culture medium of thecells cultured in (e).

The present invention provides a method for producing an antigen-bindingdomain, which comprises steps (a) to (f) below:

(a) contacting an antigen with a library of antigen-binding domains inthe presence of a high concentration of a small molecule compound;(b) obtaining antigen-binding domains that bind to the antigen in step(a);(c) placing the antigen-binding domains obtained in step (b) in thepresence of a low concentration of the compound;(d) isolating an antigen-binding domain whose antigen-binding activityin step (c) is weaker than the reference selected in step (b);(e) culturing cells transfected with a vector to which a polynucleotideencoding the antigen-binding domain selected in (d) is operably linked;and(f) collecting an antigen-binding domain from a culture medium of thecells cultured in (e).

The terms “cells”, “cell line”, and “cell culture” are used synonymouslyherein, and such naming may include all progenies of the cells or cellline. This way, for example, the terms “transformant” and “transformedcells” include cultures and primary target cells derived from themregardless of the number of passages. Furthermore, it is understood thatdue to intentional or accidental mutations, the DNA content is notalways exactly the same in all progenies. Progenies of mutants havingsubstantially the same function or biological activity such as thosescreened for in the initially transformed cells may also be included.When the description is intended to refer to a different naming, thatintention may become obvious from the context of the description. Cellsthat are appropriate for use are suitably selected from cells describedin the section “Antibodies” above.

When referring to the expression of a coding sequence, the term “controlsequences” refers to DNA nucleotide sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotesinclude, for example, a promoter, optionally an operator sequence, aribosome binding site, and possibly, other as yet poorly understoodsequences. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers for the expression of a codingsequence.

For a nucleic acid, the term “operably linked” means that the nucleicacid is placed into a functional relationship with another nucleic acidsequence. For example, DNA for a presequence or secretory leader isoperably linked to DNA for a polypeptide if it is expressed as aprecursor protein that participates in the secretion of the polypeptide.A promoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence. A ribosome binding site isoperably linked to a coding sequence if it is positioned so as tofacilitate translation. Generally, “operably linked” means that the DNAsequences being linked are contiguous and, in the case of a secretoryleader, contiguous and in reading frame. However, enhancers do not haveto be contiguous. Linking is accomplished by ligation at suitablerestriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accordance withconventional practice. Furthermore, linked nucleic acids may be producedby the above-mentioned overlap extension PCR technique.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo nucleic acid fragments. For ligation of the two fragments, the endsof the fragments must be compatible with each other. In some cases, theends will be directly compatible after endonuclease digestion. However,it may be necessary first to convert the staggered ends commonlyproduced after endonuclease digestion to blunt ends to make themcompatible for ligation. For blunting the ends, the DNA is treated in asuitable buffer for at least 15 minutes at 15° C. with about 10 units ofthe Klenow fragment of DNA polymerase I or T4 DNA polymerase in thepresence of the four deoxyribonucleotide triphosphates. The DNA is thenpurified by phenol-chloroform extraction and ethanol precipitation, orby silica purification. The DNA fragments that are to be ligatedtogether are put in solution in equimolar amounts. The solution willcontain ATP, ligase buffer, and a ligase such as T4 DNA ligase at about10 units per 0.5 μg of DNA. If the DNA is to be ligated into a vector,the vector is first linearized by digestion with the appropriaterestriction endonuclease(s). The linearized fragment is then treatedwith bacterial alkaline phosphatase or calf intestinal phosphatase toprevent self-ligation of the fragment during the ligation step.

In the production methods of the present invention, an antigen-bindingdomain which has a higher antigen-binding activity in the presence of asmall molecule compound than in its absence, which has been selected bythe method described in the above section “Antigen-binding domaindependent on a small molecule compound” is isolated. For example, whenan antigen-binding domain isolated in this manner has been selected froma library, the polynucleotide encoding the antigen-binding domain isisolated by general gene amplification from a virus such as a phage, asdescribed in the Examples below. Furthermore, when an antigen-bindingdomain or an antibody isolated in this manner has been selected fromculture media of cells such as hybridomas, the antibody gene or such canbe isolated by general gene amplification from the cells as shown in thesection “Antibodies” above.

Methods for Producing Antigen-Binding Molecules

The present invention provides methods for producing antigen-bindingmolecules whose antigen-binding activity in the presence of a smallmolecule compound is higher than the antigen-binding activity in theabsence of the compound.

More specifically, the present invention provides a method for producingantigen-binding molecules, which comprises the steps of:

(a) determining the antigen-binding activity of an antigen-bindingdomain in the absence of a small molecule compound;

(b) determining the antigen-binding activity of the antigen-bindingdomain in the presence of the small molecule compound;

(c) selecting an antigen-binding domain with lower antigen-bindingactivity in the absence of the small molecule compound than in thepresence of the compound;

(d) linking a polynucleotide encoding the antigen-binding domainselected in (c) to a polynucleotide encoding a polypeptide containing anFc region;

(e) culturing cells introduced with a vector to which the polynucleotideobtained in (d) is operably linked; and

(f) collecting antigen-binding molecules from a culture medium of thecells cultured in (e).

The present invention also provides a method for producing anantigen-binding molecule, which comprises the steps of:

(a) determining the antigen-binding activity of an antigen-bindingdomain in the presence of a low concentration of a small moleculecompound;

(b) determining the antigen-binding activity of the antigen-bindingdomain in the presence of a high concentration of the small moleculecompound;

(c) selecting an antigen-binding domain with lower antigen-bindingactivity in the presence of a low concentration of the small moleculecompound than in the presence of a high concentration of the compound;

(d) linking a polynucleotide encoding the antigen-binding domainselected in (c) to a polynucleotide encoding a polypeptide containing anFc region;

(e) culturing cells introduced with a vector to which the polynucleotideobtained in (d) is operably linked; and

(f) collecting antigen-binding molecules from a culture medium of thecells cultured in (e).

Furthermore, the present invention provides a method for producing anantigen-binding molecule, which comprises the steps of:

(a) contacting antigen-binding domains or a library thereof with anantigen in the presence of a small molecule compound;

(b) placing the antigen-binding domains that bind to the antigen in saidstep (a) in the absence of the compound;

(c) isolating an antigen-binding domain that dissociates in said step(b);

(d) linking a polynucleotide encoding the antigen-binding domainselected in (c) to a polynucleotide encoding a polypeptide containing anFc region;

(e) culturing cells introduced with a vector to which the polynucleotideobtained in (d) is operably linked; and

(f) collecting an antigen-binding molecule from a culture medium of thecells cultured in (e).

In addition, the present invention provides a method for producing anantigen-binding molecule, which comprises the steps of:

(a) contacting antigen-binding domains or a library thereof with anantigen in the presence of a high concentration of a small moleculecompound;

(b) placing the antigen-binding domains that bind to the antigen in saidstep (a) in the presence of a low concentration of the compound;

(c) isolating an antigen-binding domain that dissociates in said step(b);

(d) linking a polynucleotide encoding the antigen-binding domainselected in (c) to a polynucleotide encoding a polypeptide containing anFc region;

(e) culturing cells introduced with a vector to which the polynucleotideobtained in (d) is operably linked; and

(f) collecting antigen-binding molecules from a culture medium of thecells cultured in (e).

The present invention provides a method for producing an antigen-bindingmolecule, which comprises the steps of:

(a) contacting a library of antigen-binding domains with an antigen inthe absence of a small molecule compound;

(b) selecting antigen-binding domains that do not bind to the antigen insaid step (a);

(c) allowing the antigen-binding domains selected in said step (b) tobind to the antigen in the presence of the compound;

(d) isolating an antigen-binding domain that binds to the antigen insaid step (c);

(e) linking a polynucleotide encoding the antigen-binding domainselected in (d) to a polynucleotide encoding a polypeptide containing anFc region;

(f) culturing cells introduced with a vector to which the polynucleotideobtained in (e) is operably linked; and

(g) collecting antigen-binding molecules from a culture medium of thecells cultured in (0.

The present invention provides a method for producing an antigen-bindingmolecule, which comprises the steps of:

(a) contacting a library of antigen-binding domains with an antigen inthe presence of a low concentration of a small molecule compound;

(b) selecting antigen-binding domains that do not bind to the antigen insaid step (a);

(c) allowing the antigen-binding domains selected in said step (b) tobind to the antigen in the presence of a high concentration of thecompound;

(d) isolating an antigen-binding domain that binds to the antigen insaid step (c);

(e) linking a polynucleotide encoding the antigen-binding domainselected in (d) to a polynucleotide encoding a polypeptide containing anFc region;

(f) culturing cells introduced with a vector to which the polynucleotideobtained in (e) is operably linked; and

(g) collecting antigen-binding molecules from a culture medium of thecells cultured in (0.

The present invention provides a method for producing an antigen-bindingmolecule, which comprises the steps of:

(a) contacting a library of antigen-binding domains with anantigen-immobilized column in the presence of a small molecule compound;

(b) eluting antigen-binding domains that bind to the column in said step(a) from the column in the absence of the compound;

(c) isolating an antigen-binding domain eluted in said step (b);

(d) linking a polynucleotide encoding the antigen-binding domainselected in (c) to a polynucleotide encoding a polypeptide containing anFc region;

(e) culturing cells introduced with a vector to which the polynucleotideobtained in (d) is operably linked; and

(f) collecting antigen-binding molecules from a culture medium of thecells cultured in (e).

The present invention provides a method for producing an antigen-bindingmolecule, which comprises the steps of:

(a) contacting a library of antigen-binding domains with anantigen-immobilized column in the presence of a high concentration of asmall molecule compound;

(b) eluting antigen-binding domains that bind to the column in said step(a) from the column in the presence of a low concentration of thecompound;

(c) isolating an antigen-binding domain eluted in said step (b);

(d) linking a polynucleotide encoding the antigen-binding domainselected in (c) to a polynucleotide encoding a polypeptide containing anFc region;

(e) culturing cells introduced with a vector to which the polynucleotideobtained in (d) is operably linked; and

(f) collecting antigen-binding molecules from a culture medium of thecells cultured in (e).

The present invention provides a method for producing an antigen-bindingmolecule, which comprises the steps of:

(a) allowing a library of antigen-binding domains to pass through anantigen-immobilized column in the absence of a small molecule compound;

(b) collecting antigen-binding domains that are eluted without bindingto the column in said step (a);

(c) allowing the antigen-binding domains collected in step (b) to bindto the antigen in the presence of the compound;

(d) isolating an antigen-binding domain that binds to the antigen instep (c);

(e) linking a polynucleotide encoding the antigen-binding domainselected in (d) to a polynucleotide encoding a polypeptide containing anFc region;

(f) culturing cells introduced with a vector to which the polynucleotideobtained in (e) is operably linked; and

(g) collecting antigen-binding molecules from a culture medium of thecells cultured in (f).

The present invention provides a method for producing an antigen-bindingmolecule, which comprises the steps of:

(a) allowing a library of antigen-binding domains to pass through anantigen-immobilized column in the presence of a low concentration of asmall molecule compound;

(b) collecting antigen-binding domains that are eluted without bindingto the column in said step (a);

(c) allowing the antigen-binding domains collected in said step (b) tobind to the antigen in the presence of a high concentration of thecompound;

(d) isolating an antigen-binding domain that binds to the antigen insaid step (c);

(e) linking a polynucleotide encoding the antigen-binding domainselected in (d) to a polynucleotide encoding a polypeptide containing anFc region;

(f) culturing cells introduced with a vector to which the polynucleotideobtained in (e) is operably linked; and

(g) collecting antigen-binding molecules from a culture medium of thecells cultured in (f).

Furthermore, the present invention provides a method for producing anantigen-binding molecule, which comprises the steps of:

(a) contacting a library of antigen-binding domains with an antigen inthe presence of a small molecule compound;

(b) obtaining antigen-binding domains that bind to the antigen in saidstep (a);

(c) placing the antigen-binding domains obtained in said step (b) in theabsence of the compound;

(d) isolating an antigen-binding domain whose antigen-binding activityin said step (c) is weaker than the reference selected in step (b);

(e) linking a polynucleotide encoding the antigen-binding domainselected in (d) to a polynucleotide encoding a polypeptide containing anFc region;

(f) culturing cells introduced with a vector to which the polynucleotideobtained in (e) is operably linked; and

(g) collecting antigen-binding molecules from a culture medium of thecells cultured in (0.

The present invention provides a method for producing an antigen-bindingmolecule, which comprises the steps of:

(a) contacting a library of antigen-binding domains with an antigen inthe presence of a high concentration of a small molecule compound;

(b) obtaining antigen-binding domains that bind to the antigen in saidstep (a);

(c) placing the antigen-binding domains obtained in step (b) in thepresence of a low concentration of the compound;

(d) isolating an antigen-binding domain whose antigen-binding activityin step (c) is weaker than the reference selected in step (b);

(e) linking a polynucleotide encoding the antigen-binding domainselected in (d) to a polynucleotide encoding a polypeptide containing anFc region;

(f) culturing cells introduced with a vector to which the polynucleotideobtained in (e) is operably linked; and

(g) collecting antigen-binding molecules from a culture medium of thecells cultured in (f).

The present invention also provides a method for producing anantigen-binding molecule that comprises an antigen-binding domain whoseantigen-binding activity varies depending on the concentration of asmall molecule compound, which comprises the steps of:

(a) contacting a library of the present invention with an antigen in theabsence of a small molecule compound;

(b) selecting an antigen-binding domain that does not bind to theantigen in step (a) above;

(c) contacting the antigen-binding domain selected in step (b) abovewith the antigen in the presence of the small molecule compound;

(d) selecting an antigen-binding domain that binds to the antigen instep (c) above;

(e) linking a polynucleotide encoding the antigen-binding domainselected in step (d) above with a polynucleotide encoding a polypeptidecomprising an Fc region;

(f) culturing cells introduced with a vector in which the polynucleotideobtained in step (e) above is operably linked; and

(g) collecting the antigen-binding molecule from the culture solution ofcells cultured in step (0 above.

The present invention also provides a method for producing anantigen-binding molecule that comprises an antigen-binding domain whoseantigen-binding activity varies depending on the concentration of asmall molecule compound, which further comprises in addition to theabove-mentioned embodiment the steps of:

(a) contacting a library of the present invention with a small moleculecompound; and

(b) selecting an antigen-binding domain collected in step (a) above.

Further, the present invention provides a method for producing anantigen-binding molecule that comprises an antigen-binding domain whoseantigen-binding activity varies depending on the concentration of asmall molecule compound, which comprises the steps of:

(a) contacting a library of the present invention with an antigen in thepresence of a small molecule compound;

(b) collecting an antigen-binding domain by dissociation using a lowerconcentration of the small molecule compound than in step (a) above;

(c) linking a polynucleotide encoding the antigen-binding domaincollected in step (b) above with a polynucleotide encoding a polypeptidecomprising an Fc region;

(d) culturing cells introduced with a vector in which the polynucleotideobtained in step (c) above is operably linked; and

(e) collecting an antigen-binding molecule from the culture solution ofcells cultured in step (d) above.

The present invention also provides a method for producing anantigen-binding molecule that comprises an antigen-binding domain whoseantigen-binding activity varies depending on the concentration of asmall molecule compound, which further comprises in addition to theabove-mentioned embodiment, the steps of:

(a) contacting a library of the present invention with the smallmolecule compound; and

(b) selecting an antigen-binding domain collected in step (a) above.

A non-limiting embodiment of the Fc region whose polynucleotide sequenceis linked to a polynucleotide encoding an antigen-binding domain is, forexample, the Fc region contained in the constant region of a human IgG1(SEQ ID NO: 5), IgG2 (SEQ ID NO: 6), IgG3 (SEQ ID NO: 7), or IgG4 (SEQID NO: 8) antibody. An Fc region is a portion of the heavy chainconstant region of an antibody, starting from the N terminal end of thehinge region, which corresponds to the papain cleavage site at an aminoacid around position 216 according to EU numbering, and contains thehinge, CH2, and CH3 domains. The Fc region may be obtained from humanIgG1, but it is not limited to any particular subclass of IgG.

A non-limiting embodiment of the Fc region whose polynucleotide sequenceis linked to a polynucleotide encoding an antigen-binding domainincludes, for example, Fc regions whose binding activity toward anactivating FcγR is lower than that of the native Fc region toward anactivating FcγR. Another non-limiting embodiment of the Fc regionpreferably includes, for example, Fc regions in which one or more aminoacids at positions 234, 235, 236, 237, 238, 239, 270, 297, 298, 325,328, and 329 according to EU numbering are altered to amino acids thatare different from those of the native Fc region of SEQ ID NO: 5, 6, 7,or 8. The alterations in the Fc region are not limited to the aboveexample, and they may be, for example, alterations such asdeglycosylation (N297A and N297Q), IgG1-L234A/L235A,IgG1-A325A/A330S/P331S, IgG1-C226S/C229S,IgG1-C226S/C229S/E233P/L234V/L235A, IgG1-L234F/L235E/P331S,IgG1-S267E/L328F, IgG2-V234A/G237A, IgG2-H268Q/V309L/A330S/A3315,IgG4-L235A/G237A/E318A, and IgG4-L236E described in Cur. Opin. inBiotech. (2009) 20 (6), 685-691; alterations such as G236R/L328R,L235G/G236R, N325A/L328R, and N325L/L328R described in WO 2008/092117;amino acid insertions at positions 233, 234, 235, and 237 according toEU numbering; and alterations at the positions described in WO2000/042072.

When the Fc region contained in the antigen-binding molecule of thepresent invention is an Fc region that has been modified so that thepercentage of the Fc region to which a fucose-deficient sugar chain hasbeen attached, or bisecting N-acetylglucosamine has been attached, willbecome higher, the above-mentioned transformed host cells that aresuitably used are host cells that have low ability to add fucose to asugar chain as a result of modification of the activity to form thesugar chain structure of a polypeptide to be modified with a sugar chain(for example, WO 2000/061739, WO 2002/031140, and WO 2006/067913). In anon-limiting embodiment of such host cells, host cells deficient in theactivity of an enzyme or transporter selected from the group consistingof fucosyltransferase (EC 2.4.1.152), fucose transporter (SLC35C1), GMD(GDP-mannose-4,6-dehydratase) (EC 4.2.1.47), Fx(GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase) (EC 1.1.1.271), andGFPP (GDP-β-L-fucose pyrophosphorylase (EC 2.7.7.30), may be suitablyused (for example, WO 2000/061739, WO 2002/031140, and WO 2006/067913).Host cells deficient in such activity can be produced, for example, by amethod that destroys the genes of these functional proteins endogenousto CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63mouse myeloma cells, PER cells, PER.C6 cells, HEK293 cells, hybridomacells, or such so that they are unable to function.

When the Fc region contained in the antigen-binding molecule of thepresent invention is an Fc region having a sugar chain containing abisecting GlcNAc, the above-described transformed cells that aresuitably used are host cells expressing a gene encoding a functionalprotein having GnTIII (β-1,4-mannosyl-glycoprotein4-β-N-acetylglucosaminyltransferase) (EC2.4.1.144) activity or GalT(β-1,4-galactosyltransferase) (EC 2.4.1.38) activity to produceantibodies which have bisecting GlcNAc-containing sugar chains(WO2002/079255 and such). In another suitable non-limiting embodiment,host cells that co-express, in addition to the aforementioned functionalproteins, a gene encoding a functional protein having human ManII(mannosidase II) (3.2.1.114) activity, a gene encoding a functionalprotein having GnTI (β-1,2-acetylglucosaminyltransferase I) (EC2.4.1.94) activity, a gene encoding a functional protein having GnTII(β-1,2-acetylglucosaminyltransferase II) (EC 2.4.1.143) activity, a geneencoding a functional protein having ManI (mannosidase) (EC 3.2.1.113)activity, and α-1,6-fucosyl transferase (EC 2.4.1.68), are suitably used(WO2004/065540).

Antigen-binding molecules of the present invention are produced usingmethods that follow the methods for producing antibodies, such asisolation from culture media of the above-mentioned cells, which aredescribed in the section “Antibodies” above. A non-limiting embodimentof the aforementioned polypeptides containing an Fc region includes, forexample, the antibody constant region of SEQ ID NO: 5, 6, 7, or 8. Anon-limiting embodiment of the antigen-binding molecules of the presentinvention is for example, a full-length antibody molecule.

Pharmaceutical Compositions

The present invention provides pharmaceutical compositions comprising anantigen-binding molecule that does not act systemically in the blood ornormal tissues, but acts on lesions such as cancer and inflamed sites,to exhibit drug efficacy while avoiding side effects. The binding of theantigen-binding molecule contained in the pharmaceutical composition ofthe present invention to target antigen is regulated depending on theconcentration of target tissue-specific compounds that are specificallypresent or produced in a target tissue and/or unnatural compounds thataccumulate in the tissue. Thus, for example, when the antigen-bindingmolecule targets an antigen in a cancer tissue or inflammatory tissue,it binds to an antigen expressed in cancer cells, immune cells, stromalcells, or such in cancer tissues; an antigen secreted in cancer tissues;or an antigen expressed by immune cells or such in inflammatory tissues;and an antigen secreted in inflammatory tissues; and cannot bind toantigens expressed in normal tissues; therefore, side effects due tocytotoxic activity, neutralizing activity, or such against normaltissues are avoided; and at the same time, potent cytotoxic effects,growth suppressing effects, and immunity-enhancing action on cancers, orimmunosuppressive effects against inflammatory cells in inflammatorytissues, are exhibited. For example, a bispecific or biparatopicantigen-binding molecule containing an antigen-binding domain that bindsto EGFR expressed on cancer cells and an antigen-binding domain thatbinds to CD3 expressed on T cells in a manner dependent on a cancertissue-specific compound, does not bind to EGFR expressed on normaltissues but bind to EGFR expressed on cancer cells; thereby exhibitingpotent antitumor effects while avoiding side effects. Specifically,while the antigen-binding molecule binds to CD3 expressed on T cells inthe vicinity of cancer cells in a manner dependent on a cancertissue-specific compound, the molecule does not bind to CD3 expressed onT cells that are not in the vicinity of cancer cells. Therefore, themolecule activates T cells in the vicinity of cancer cells, exhibitingpotent antitumor effects while avoiding side effects.

Such antigen-binding molecules that bind to an antigen in target tissuesbut not in other normal tissues and blood exhibit drug efficacy whileavoiding side effects. Antigen-binding molecules provided by the presentinvention, which bind to an antigen by using a small molecule present athigh concentrations in target tissues in vivo as a switch, namely, smallmolecule switch antigen-binding molecules, do not bind to the antigen ina normal environment where the small molecule is not present, but canbind to the antigen in target tissues where the small molecule ispresent at high concentrations.

A non-limiting embodiment of such small molecule switch antigen-bindingmolecules includes cancer tissue-specific compound-dependent orinflammatory tissue-specific compound-dependent antigen-bindingmolecules, in which a cancer tissue-specific or inflammatorytissue-specific compound such as adenosine, adenosine 5′-triphosphate(ATP), inosine, kynurenine, prostaglandin E2 (PGE2), succinic acid, andlactic acid, which are present at a high concentration in cancer tissuesor inflammatory tissues and capable of functioning as a switch, providesa switch function by being sandwiched between the antigen-bindingmolecule of the present invention (the paratope contained therein) andthe antigen (the epitope contained therein), or by binding with theantigen-binding molecule of the present invention to thereby change thestructure of the paratope of the antigen-binding molecule for theantigen. In the absence of the compound, the interaction between theparatope in the antigen-binding molecule of the present invention andthe epitope in the antigen is not sufficient for the antigen-bindingmolecule of the present invention to be able to bind to the antigen. Inthe presence of the compound, the compound interposes between theparatope in the antigen-binding molecule of the present invention andthe epitope in the antigen, or changes the structure of the paratope;and the antigen-binding molecule that has bound to the antigen in atarget tissue such as cancer tissue or inflammatory tissue, where thecompound is present at a high concentration, can exhibit drug efficacyon cells expressing the antigen. Moreover, since this binding of theswitch compound is reversible, the binding of an antigen-bindingmolecule of the present invention to an antigen by means of these switchcompounds may be controlled in a reversible manner. Thus,antigen-binding molecules of the present invention which can exhibitdrug efficacy in a lesion site such as cancer tissue or inflammatorytissue by binding to pathogenic cells such as cancer cells or immunecells in a cancer tissue or inflammatory tissue or by binding to anantigen secreted in a cancer tissue or inflammatory tissue are useful aspharmaceutical compositions. The pharmaceutical compositions of thepresent invention may comprise a pharmaceutically acceptable carrier.

In the present invention, pharmaceutical compositions generally refer topharmaceutical agents for treating or preventing, or testing anddiagnosing diseases. Furthermore, in the present invention, the phrase“pharmaceutical composition containing an antigen-binding molecule whoseantigen-binding activity varies depending on the concentration of asmall molecule compound” (in this regard, a small molecule compoundincludes a target tissue-specific compound, unnatural compound, andsuch) can be rephrased as “method for treating a disease which comprisesadministering to a subject to be treated an antigen-binding moleculewhose antigen-binding activity varies depending on the concentration ofa small molecule compound”, or rephrased as “use of an antigen-bindingmolecule whose antigen-binding activity varies depending on theconcentration of a small molecule compound in the production of apharmaceutical for treating a disease”. Furthermore, the phrase“pharmaceutical composition containing an antigen-binding molecule whoseantigen-binding activity varies depending on the concentration of asmall molecule compound” can be rephrased as “use of an antigen-bindingmolecule whose antigen-binding activity varies depending on theconcentration of a small molecule compound, for treating a disease”.

The pharmaceutical compositions of the present invention can beformulated by methods known to those skilled in the art. For example,they can be used parenterally, in the form of injections of sterilesolutions or suspensions including water or other pharmaceuticallyacceptable liquid. For example, such compositions can be formulated bymixing in the form of unit dose required in the generally approvedmedicine manufacturing practice, by appropriately combining withpharmacologically acceptable carriers or media, specifically withsterile water, physiological saline, vegetable oil, emulsifier,suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle,preservative, binder, or such. In such formulations, the amount ofactive ingredient is adjusted to obtain an appropriate amount in apre-determined range.

Sterile compositions for injection can be formulated using vehicles suchas distilled water for injection, according to standard formulationpractice. Aqueous solutions for injection include, for example,physiological saline and isotonic solutions containing dextrose or otheradjuvants (for example, D-sorbitol, D-mannose, D-mannitol, and sodiumchloride). It is also possible to use in combination appropriatesolubilizers, for example, alcohols (ethanol and such), polyalcohols(propylene glycol, polyethylene glycol, and such), non-ionic surfactants(polysorbate 80™, HCO-50, and such).

Oils include sesame oil and soybean oils. Benzyl benzoate and/or benzylalcohol can be used in combination as solubilizers. It is also possibleto combine buffers (for example, phosphate buffer and sodium acetatebuffer), soothing agents (for example, procaine hydrochloride),stabilizers (for example, benzyl alcohol and phenol), and/orantioxidants. Appropriate ampules are filled with the preparedinjections.

The pharmaceutical compositions of the present invention are preferablyadministered parenterally. For example, the compositions in the dosageform for injections, transnasal administration, transpulmonaryadministration, or transdermal administration are administered. Forexample, they can be administered systemically or locally by intravenousinjection, intramuscular injection, intraperitoneal injection,subcutaneous injection, or such.

Administration methods can be appropriately selected in consideration ofthe patient's age and symptoms. The dose of a pharmaceutical compositioncontaining an antigen-binding molecule can be, for example, from 0.0001to 1,000 mg/kg for each administration. Alternatively, the dose can be,for example, from 0.001 to 100,000 mg per patient. However, the presentinvention is not limited by the numeric values described above. Thedoses and administration methods vary depending on the patient's weight,age, symptoms, and such. Those skilled in the art can set appropriatedoses and administration methods in consideration of the factorsdescribed above.

Amino acids contained in the amino acid sequences of the presentinvention may be post-translationally modified (for example, themodification of an N-terminal glutamine into a pyroglutamic acid bypyroglutamylation is well-known to those skilled in the art). Naturally,such post-translationally modified amino acids are included in the aminoacid sequences in the present invention.

Those skilled in the art will naturally understand that any arbitrarycombination of one or more of the embodiments described herein areincluded in the present invention, as long as it is not technicallyinconsistent with the common general knowledge of those skilled in theart.

All prior art documents cited in this specification are incorporatedherein by reference.

Herein below, the present invention will be specifically described withthe Examples; however, the present invention should not be limitedthereto.

EXAMPLES Example 1 Concept of and Strategy for Obtaining SwitchAntibodies that Bind to Antigens Using Small Molecules which are Presentat High Concentrations in Target Tissues as a Switch (1-1) Concept ofSwitch Antibodies Whose Antigen-Binding Ability Varies in the Presenceof Target Tissue-Specific Compounds

In order to exert drug efficacy while avoiding adverse effects, there isa need for drug discovery technology that works in lesions such ascancer or inflammatory sites without acting systemically in normaltissues or blood. Antibody molecules that can bind to antigens expressedon cancer cells but are incapable of binding to the antigens expressedon normal tissues after administration can exert strong cytotoxiceffects against cancer while avoiding adverse effects on normal tissuesas a result of cytotoxic action. For example, antigen-binding moleculesthat have been altered from the above-described EGFR-BiTE (Non-patentDocument 9), which cannot bind to EGFR expressed on normal tissues butare capable of binding to EGFR expressed on cancer cells, can exertstrong an antitumor effect while avoiding adverse effects. Meanwhile,BiTE exerts an antitumor effect by recruiting and activating T cells viaCD3 (Non-patent Document 8); and if it is possible to confer EGFR-BiTEwith the property of binding to CD3 expressed on T cells in the vicinityof cancer cells but not to CD3 expressed on T cells outside the vicinityof cancer cells, EGFR-BiTE altered to have the property can activate Tcells in cancer and thus can exert strong antitumor effects whileavoiding adverse effects.

However, this is not limited to only antibody pharmaceuticals againstcancer. When an antibody molecule binds and inhibits cytokines in thesynovial fluid of inflamed joints in rheumatoid arthritis but does notsystemically inhibit the cytokines, the molecule can exert potenttherapeutic effects against inflammatory/autoimmune diseases such asrheumatoid arthritis while avoiding increased risks of infection due tosystemic neutralization of cytokines.

As described above, antibodies that bind to antigens in cancer tissuesbut not to antigens in other tissues such as normal tissues and bloodcan exert drug efficacy while avoiding adverse effects. However, idealantibodies having such properties have not been reported so far.Meanwhile, as shown in FIG. 1, antibody molecules that bind to antigensvia small molecules, as a switch, that are present at highconcentrations in cancer tissues in vivo or via compounds that have aproperty of accumulating in cancer tissues after being administered invivo (i.e., small molecule switch antibodies), do not bind to antigensin environments in the absence of such small molecules; and they canbind to antigens in target tissues where the small molecules are presentat high concentrations.

In developing such small-molecule switch antibodies, first it was tosearch for small molecules that are present at high concentration incancer tissues and are considered to be usable as a switch. The resultsuggested that adenosine, adenosine triphosphate (adenosine5′-triphosphate (ATP)), inosine, kynurenine, prostaglandin E2 (PGE2),succinic acid, and lactic acid were promising as a switch. Each of thesesmall molecules is either produced by cancer cells, or released fromcancer cells after cell death, or produced by immune cells etc.infiltrating cancer tissues, and thus they are present at highconcentrations in cancer tissues; however, they are present at lowerconcentrations in normal tissues and blood in comparison to cancertissues.

Next, a search was carried out for molecules having the property toaccumulate in cancer tissues following in vivo administration. Prodrugssuch as Xeloda and TH302 when administered in vivo are metabolized bymetabolic enzymes expressed in cancer tissues and produce smallmolecules that can serve as a switch. Thus, 5-fluorouracil (5-FU),Br-IPM, and such were expected to be useful as a switch. 5-FU is ametabolic product of Capecitabine (Xeloda) and is known to bemetabolized by the cancer tissue-specific metabolic enzymes cytidinedeaminase and thymidine phosphorylase (Desmoulin F. et al., Drug MetabDispos. 2002). Meanwhile, TH-302 is known to be converted into Br-IPM byreduction under hypoxic conditions such as around cancer tissues (Duan JX, et al., J Med Chem. 2008). Thus, after in vivo administration, theprodrugs are thought to be metabolized by metabolic enzymes expressed incancer tissues and to exist at high concentrations, while in normaltissues and blood, they are thought to exist at low concentrations ascompared to in cancer tissues.

If these small molecules can be sandwiched in the complex between theantigen and the antibody as shown in FIG. 2, the molecules could fulfillthe function as a switch. Alternatively, if these small molecules canalter the antigen-binding ability of an antibody by binding to it andchanging the conformation of the antigen-binding site of the antibody,these small molecules could fulfill the function as a switch.Specifically, in the absence of the small molecules, the interactionbetween the antigen and the antibody is insufficient and the antibodycannot bind to the antigen; however, in the presence of the smallmolecules, the antibody can bind to the antigen. In other words, in thepresence of a low concentration of the small molecules, the interactionbetween the antigen and the antibody is insufficient and the antibodycannot bind to the antigen; however, in the presence of a highconcentration of the small molecules, the antibody can bind to theantigen. Furthermore, since the binding of the small molecules thatbecome a switch is reversible, the regulation of antigen binding bythese small molecule switches is reversible.

Alternatively, the action of the antibody can be regulated throughadministration of an oral agent, by oral administration of an exogenouscompound serving as a switch. Specifically, when a switch antibody thatbinds to an antigen using an exogenous compound as a switch, whichcompound can be administered non-invasively such as by oraladministration, is invasively administered e.g. intravenously orsubcutaneously, the action of the antibody can be regulated bynon-invasively administering the exogenous compound that becomes aswitch by oral administration or such. Antibody pharmaceuticals havelong half-lives; thus, if adverse effects occur, the effect will beprolonged, and this is a disadvantage. However, if the action of theantibody can be regulated in this way by non-invasively administeringexogenous compounds such as by oral administration, the action of thepharmaceutical can be discontinued by interrupting the administration ofthe switch molecule when adverse effects occurred. Moreover, bypreliminarily administering a switch antibody, pharmacological effectscould be exerted only when necessary by non-invasive administration suchas oral administration by administering switch molecules only whensymptoms occurred due to the disease.

(1-2) Strategy for Obtaining Switch Antibodies Whose Antigen-BindingAbility Varies in the Presence of Target Tissue-Specific Compounds

Methods for more efficiently producing switch antibodies that bind toantigens in a reversible manner depending on the presence of targettissue-specific compounds include methods that use library techniques.When, using as a template an antibody that maintains binding with atissue-specific compound, its variable region that is not involved inthe binding with the compound is made into a library, antibodies capableof binding to the compound appear at a higher frequency than in ordinaryantibody libraries, suggesting that antigen-binding molecules havingdesired properties could be obtained efficiently. Thus, to first obtainan antibody for use as template sequence for the library, acquisition ofantibodies that bind to adenosine or ATP, which are known to be presentat high concentrations in cancer cells, was attempted.

Example 2 Acquisition of Anti-Adenosine Antibodies by Rabbit B CellCloning (2-1) Design of Immunogen to Construct Adenosine-Binding Library

The immunogens used in immunizing rabbits were 2′-Adenosine-PEG-Tetanustoxin p30 helper peptide (2′-Adenosine-PEG-peptide) shown in FIG. 3 and5′-Adenosine-PEG-Tetanus toxin p30 helper peptide(5′-Adenosine-PEG-peptide) shown in FIG. 4. The Tetanus toxin p30 helperpeptide consists of the amino acid sequence FNNFTVSFWLRVPKVSASHLE (SEQID NO: 4), and is a peptide identified as an epitope of T cell receptorexpressed on helper T cells (Eur. J. Immunol. (1989) 19, 2237-2242). Thepeptide is known to activate antibody production (J. Immunol. (1992)149, 717-721). When linked to adenosine, the peptide serves as anadjuvant and thus is expected to enhance the production of antibodiesagainst adenosine. The linkage between adenosine and the Tetanus toxinp30 helper peptide was designed to be through PEG so that epitopes ofantibodies against adenosine can hardly contain the Tetanus toxin p30helper peptide. Adenosine is an ATP metabolite, and since the phosphategroups of ATP are attached to the 5′ hydroxyl group of adenosine,antibodies that do not recognize the 5′ hydroxyl group of adenosine asan epitope may also bind to ATP in addition to adenosine. That is, itwould be easier to obtain antibodies that can bind to both adenosine andATP by using as an immunogen the 5′-Adenosine-PEG-Tetanus toxin p30helper peptide, while it would be easier to obtain antibodies that bindto adenosine but not to ATP by using as an immunogen the2′-Adenosine-PEG-Tetanus toxin p30 helper peptide. For this reason, thetwo types of immunogens which contain the Tetanus toxin p30 helperpeptide linked to the 2′ or 5′ position of adenosine were prepared inthe manner described in (2-2).

In addition, 2′-Adenosine-PEG-biotin (FIG. 5) and5′-Adenosine-PEG-biotin (FIG. 6), in which biotin is conjugated insteadof the Tetanus toxin p30 helper peptide, were produced as describedbelow. By assessing the binding to these two types ofAdenosine-PEG-biotin, antibodies can be tested to demonstrate that theirepitopes do not contain the Tetanus toxin p30 helper peptide.

(2-2) Synthesis of Immunogens to Prepare Adenosine-Binding Library

2′-Adenosine-PEG-peptide (adenosine 2′-PEG-peptide conjugate or2′-(PEG-peptide)adenosine) and 2′-Adenosine-PEG-biotin (adenosine2′-PEG-biotin conjugate or 2′-(PEG-biotin)adenosine) were synthesized inthe manner described below. The synthesized 2′-Adenosine-PEG-peptide and2′-Adenosine-PEG-biotin were analyzed or fractionated under theconditions below.

The conditions of LCMS analysis are noted as below.

TABLE 1 Column Analysis Column Flow rate temperature condition Apparatus(length, mm) Mobile phase Gradient (A/B) (ml/min) (C.°) WavelengthSQDAA05 Acquity Aldrich Ascentis A) 10 mM AcONH4, H2O 95/5 => 0/100(1.0min) => 1.0 35 210-400 nm UPLC/SQD Express C18 B) MeOH 0/100(0.4 min)PDA total (2.1 × 50) SQDAA50 Acquity Aldrich Ascentis A) 10 mM AcONH4,H2O 50/50 => 0/100 (0.7 min) => 1.0 35 210-400 nm UPLC/SQD Express C18B) MeOH 0/100(0.7 min) PDA total (2.1 × 50) SQDFA05 Acquity AldrichAscentis A) 0.1% FA, H2O 95/5 => 0/100(1.0 min) => 1.0 35 210-400 nmUPLC/SQD Express C18 B) 0.1% FA CH3CN 0/100(0.4 min) PDA total (2.1 ×50) SQDFA50 Acquity Aldrich Ascentis A) 0.1% FA, H2O 50/50 => 0/100 (0.7min) => 1.0 35 4210-400 nm UPLC/SQD Express C18 B) 0.1% FA CH3CN0/100(0.7 min) PDA total (2.1 × 50)

The conditions of preparative HPLC are described as below.

TABLE 2 Flow Column Preparative Column (length, rate temperaturecondition Apparatus mm) Mobile phase Gradient (A/B) (ml/min) (C. °)Wavelength A Preparative HPLC Aldrich Ascentis A) 0.1% FA H2Oisocratic(A/B): 20.0 40 254, 258 nm system with injection/ RP-Amide B)0.1% FA MeCN 15/85 fractionation (21.2 × 150 (Gilson, Inc.) mm 5 μm) BPreparative HPLC YMC Actus A) 20 mM AcONH4 H2O isocratic(A/B): 20.0 40254, 258 nm system with injection/ ODS-A B) 20 mM AcONH4 MeOH/ 47/53fractionation (20 × 100 MeCN(1/1) (Gilson, Inc.) mm 5 μm)

(2-2-1) Synthesis of Compound 006 (Boc-Phe-Asn-Asn-Phe-Thr (tBu)-Val-Ser(tBu)-Phe-Trp (Boc)-Lue-Arg (Pbf)-Val-Pro-Lys (Boc)-Val-Ser(tBu)-Ala-Ser (tBu)-His (Trt)-Leu-Glu (tBu)-OH)

Peptide synthesis was performed by the Fmoc method using a peptidesynthesizer (Multipep RS; Intavis). All Fmoc amino acids were purchasedfrom WATANABE CHEMICAL INDUSTRIES, LTD. The detailed procedure of thetreatment was in the manual attached to the synthesizer.

Fmoc-Glu(tBu)-OH linked at its C terminus to 2-chlorotrityl resin (250mg/column, 30 columns, 11.7 mmol), an N,N-dimethylformamide solutioncontaining various Fmoc amino acids (0.6 mol/1) and1-hydroxy-7-azabenzotriazole (0.375 mol/1), and an N,N-dimethylformamidesolution (10% v/v) of diisopropylcarbodiimide were loaded in thesynthesizer. The synthesis reaction was performed using as anFmoc-deprotection solution, an N,N-dimethylformamide solution (20% v/v)containing piperidine and 5% (wt/v) urea. After the resin was washedwith N,N-dimethylformamide, Fmoc deprotection was carried out, followedby one cycle of Fmoc amino acid condensation reaction. This cycle wasrepeated to elongate peptides on the resin surface. After elongation,the resin was washed with trifluoroethanol. Peptides were cleaved offfrom the resin by adding trifluoroethanol/dichloromethane (=1/1). Thus,compound 006 (7.2 g) was obtained as a crude product.

LCMS(ESI) m/z=1185(M+3H)3+

Retention time: 1.24 minute (Analysis condition, SQDAA05)

(2-2-2) Synthesis of Compound 007

A suspension of adenosine (2.00 g, 7.48 mmol) in N,N-dimethylformamide(40 ml) was cooled down to 0° C., and 60% sodium hydride (0.42 g, 10.48mol) was added thereto. The reaction mixture was stirred for one hour at0° C. After adding methyl bromoacetate (0.76 ml, 8.01 mmol), theresulting reaction mixture was stirred for five hours at roomtemperature, and acetic acid (1 ml) and methanol (3 ml) were addedthereto. The reaction mixture was concentrated under reduced pressure.The resulting residue was purified by normal phase silica gel columnchromatography (dichloromethane/methanol). Thus, compound 007 (0.93 g,37%) was obtained.

LCMS(ESI) m/z=340(M+H)+

Retention time: 0.27 minute (Analysis condition, SQDFA05)

(2-2-3) Synthesis of Compound 008

t-Butyldimethylsilyl chloride (999 mg, 6.63 mol) and imidazole (722 mg,10.61 mol) were added to a pyridine solution (8 ml) of compound 007 (900mg, 2.65 mmol). The reaction mixture was stirred for four hours at roomtemperature, and extracted with ethyl acetate/water. The extractedorganic layer was washed with a saturated sodium chloride solution, anddried over anhydrous sodium sulfate. After filtration, the organic layerwas concentrated under reduced pressure. The resulting residue waspurified by normal phase silica gel column chromatography(dichloromethane/methanol). Thus, compound 008 (1.17 g, 78%) wasobtained.

LCMS(ESI) m/z=568(M+H)+

Retention time: 1.10 minute (Analysis condition, SQDFA05)

(2-2-4) Synthesis of Compound 009

Lithium hydroxide (61 mg, 2.55 mol) dissolved in water (0.17 ml) wasadded to a solution of compound 008 (290 mg, 0.511 mmol) in methanol(0.34 ml)/tetrahydrofuran (0.34 ml). The reaction mixture was stirredfor 30 minutes at room temperature. The mixture was neutralized with 1 Mhydrochloric acid, and concentrated under reduced pressure. Theconcentrated residue was extracted with ethyl acetate/water. Theresulting organic layer was washed with a saturated sodium chloridesolution, and dried over anhydrous sodium sulfate. After filtration, theorganic layer was concentrated under a reduced pressure. Thus, compound009 (319 mg, 90%) was obtained.

LCMS(ESI) m/z=552(M−H)−

Retention time: 0.97 minute (Analysis condition, SQDFA05)

(2-2-5) Synthesis of Compounds 010 and 011

1-Hydroxybenzotriazole (75 mg, 0.553 mol) and1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (106 mg,0.553 mol) were added to an N,N-dimethylformamide (1.5 ml) solution ofcompound 009 (255 mg, 0.460 mmol), and it was stirred for three minutesat room temperature. O-(2-aminoethyl)-O′-2-azidoethyl) nonaethyleneglycol (291 mg, 0.553 mmol) was added to the reaction mixture, and itwas stirred for three hours at room temperature. The reaction mixturewas concentrated under a reduced pressure, and the resulting residue waspurified by reverse phase silica gel column chromatography (aqueous 10mM ammonium acetate solution/methanol. Compounds 010 (177 mg, 42%) and011 (72 mg, 19%) were obtained.

Compound 010

LCMS(ESI) m/z=1063(M+H)+

Retention time: 0.98 minute (Analysis condition, SQDFA05)

Compound 011

LCMS(ESI) m/z=949(M+H)+

Retention time: 0.67 minute (Analysis condition, SQDFA05)

(2-2-6) Synthesis of Compound 012

10% palladium carbon (34 mg) was added to a solution of compound 010(170 mg, 0.160 mmol) in ethanol (1 ml). The reaction mixture was stirredfor two hours under hydrogen atmosphere, and again 10% palladium carbon(34 mg) was added thereto. The reaction mixture was stirred for twohours under a hydrogen atmosphere to complete the reaction. The filtrateof the reaction solution was concentrated under a reduced pressure.Compound 012 (34 mg, 95%) was obtained.

LCMS(ESI) m/z=1037(M+H)+

Retention time: 0.70 minute (Analysis condition, SQDFA05)

(2-2-7) Synthesis of Compounds 013 and 014

Compound 006 (354 mg, 0.110 mmol), 1-hydroxybenzotriazole (13 mg, 0.100mol), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride(19 mg, 0.100 mol) were added to a solution of compound 012 (86 mg,0.083 mmol) in N,N-dimethylformamide (1.5 ml), and it was stirred fortwo hours at room temperature. The filtrate of the reaction mixture waspurified by preparative condition A described in Table 2. A mixture ofcompounds 013 and 014 (72 mg) was obtained.

Compound 013

LCMS(ESI) m/z=1525(M+3H)3+, 1144(M+4H)4+

Retention time: 1.13 minute (Analysis condition, SQDAA50)

Compound 014

LCMS(ESI) m/z=1444(M+3H)3+, 1083(M+4H)4+

Retention time: 1.02 minute (Analysis condition, SQDAA50)

(2-2-8) Synthesis of 2′-Adenosine-PEG-peptide (adenosine 2′-PEG-peptideconjugate or 2′-(PEG-peptide)adenosine) (Compound 015)

Trifluoroacetic acid (16 ml), dichloromethane (8 ml), water (1.3 ml),and tetraisopropylsilane (1.3 ml) were added to the mixture of compounds013 and 014 (42 mg), and it was stirred for six hours at roomtemperature. The residue obtained by concentrating the reaction mixtureunder reduced pressure was purified by preparative condition B describedin Table 2. Thus, compound 015 (10 mg) was obtained.

LCMS(ESI) m/z=1090(M+3H)3+, 818(M+4H)4+

Retention time: 0.52 minute (Analysis condition, SQDAA50)

(2-2-9) Synthesis of Compound 016

10% palladium carbon (34 mg) was added to a solution of compound 011 (70mg, 0.074 mmol) in ethanol (1 ml), and the reaction mixture was stirredfor five hours under hydrogen atmosphere. The filtrate of the reactionmixture was concentrated under reduced pressure. Thus, compound 016 (58mg, 85%) was obtained.

LCMS(ESI) m/z=923(M+H)+

Retention time: 0.50 minute (Analysis condition, SQDFA05)

(2-2-10) Synthesis of Compound 017

D-biotin N-succinimidyl (24 mg, 0.069 mmol) and triethylamine (13 μl,0.094 mol) were added to a solution of compound 016 (58 mg, 0.063 mmol)in N,N-dimethylformamide (1 ml), and it was stirred for two hours atroom temperature. Then, after D-biotin N-succinimidyl (5 mg, 0.015 mmol)was added, the reaction was completed upon 1.5 hours of stirring at roomtemperature. The reaction mixture was purified by reverse phase silicagel column chromatography (aqueous 10 mM ammonium acetatesolution/methanol. Compound 017 (50 mg, 69%) was obtained.

LCMS(ESI) m/z=1149(M+H)+

Retention time: 1.04 minute (Analysis condition, SQDFA05)

(2-2-11) Synthesis of 2′-Adenosine-PEG-biotin (adenosine 2′-PEG-biotinconjugate or 2′-(PEG-biotin)adenosine) (Compound 018)

A solution of 1 M tetra-n-butylammonium fluoride in tetrahydrofuran (65μl, 0.065 mmol) was added to a solution of compound 017 (62 mg, 0.054mmol) in tetrahydrofuran (2 ml), and it was stirred at room temperaturefor one hour. Then, 1 M tetra-n-butylammonium fluoride intetrahydrofuran solution (20 μl, 0.020 mmol) was added, and the reactionwas completed by stirring at room temperature for one hour. The reactionmixture was concentrated under a reduced pressure, and the residue waspurified by reverse phase silica gel column chromatography (aqueous 0.1%formic acid solution/0.1% formic acid in acetonitrile). Compound 018 (12mg, 21%) was obtained.

LCMS(ESI) m/z=1035(M+H)+

Retention time: 0.71 minute (Analysis condition, SQDAA05)

Furthermore, 5′-Adenosine-PEG-peptide and 5′-Adenosine-PEG-biotin werealso synthesized by the same reaction.

(2-3) Production of Adenosine-Binding Antibodies in Animals and AntibodyScreening

Rabbits were immunized with 2′-Adenosine-PEG-peptide and/or5′-Adenosine-PEG-peptide by a conventional method. Candidates for cellswith adenosine-binding activity were selected from suspensions of cellscollected from blood of the immunized rabbits, by using autoMACS ProSeparator and FACSAria (BD) which uses Adenosine-PEG-biotin-bindingactivity and rabbit IgG expression as indicators. Then, screening wascarried out with antibodies secreted in the culture supernatants of theselected cells. In the screening, ELISA was performed to assess thepresence of binding activity to Adenosine-PEG-biotin. ELISA was alsoperformed to assess whether adenosine, when added in combination withAdenosine-PEG-biotin at a level 1000 times or more of that ofAdenosine-PEG-biotin, suppresses the binding to Adenosine-PEG-biotin.The H-chain and L-chain variable regions were isolated by PCR from cellsselected using as an indicator the presence of theAdenosine-PEG-biotin-binding activity as well as suppression of thebinding to Adenosine-PEG-biotin by adenosine added in combination withAdenosine-PEG-biotin. The obtained variable regions were expressed incombination with a human IgG1 heavy chain constant region and a humanlight chain constant region.

Example 3 Assessment of Clones Obtained by Rabbit B Cell Cloning (3-1)Assessment of Clones Obtained by Rabbit B Cell Cloning for their BindingActivity to 2′-Adenosine-PEG-Biotin

Clones obtained by rabbit B cell cloning were assessed for their bindingactivity to adenosine by the SPR method. Antigen-antibody reactionbetween the clones and 2′-Adenosine-PEG-Biotin was kinetically analyzedusing Biacore 4000 (GE Healthcare). Sensor chip CM5 (GE Healthcare) wasimmobilized with an appropriate amount of protein A/G (Invitrogen) byamine coupling. Antibodies of interest were captured by the chip. Then,after 100 nmol/l 2′-adenosine-PEG-Biotin was interacted as an analytefor 60 seconds, the dissociation of the analyte was monitored andmeasured for 60 seconds. The running buffer used was HBS-P+ (GEHealthcare). All measurements were carried out at 25° C. The analyte wasdiluted using the running buffer.

The respective antibodies were compared for their binding activity to2′-Adenosine-PEG-Biotin using as an indicator the value (N_binding_100)of dividing the amount of binding upon interaction with2′-Adenosine-PEG-Biotin by the amount of capture (RU) for each antibody,and the value (N_stability_100) of dividing the amount of dissociationof 2′-Adenosine-PEG-Biotin from each antibody for 60 seconds afterinteraction with 2′-Adenosine-PEG-Biotin by the amount of capture (RU)for each antibody. Regarding antibodies for which the amount of capturewas 1500 RU or less, their binding was not sufficiently detectable andthus they were excluded from the subjects to be tested. The result isshown in FIG. 7. The result shown in FIG. 7 demonstrates that the B cellcloning method yielded adenosine-binding clones with various affinity.

(3-2) Assessment of 2′-Adenosine-PEG-Biotin-Binding Clones for theirBinding Activity to Adenosine and ATP, and Sequence Analysis of theClones

Clones which were demonstrated to bind to 2′-Adenosine-PEG-Biotin wereassessed for their binding to adenosine and ATP by SPR and competitiveELISA.

(3-2-1) Assessment by SPR of 2′-Adenosine-PEG-Biotin-Binding Clones fortheir Binding to Adenosine

Using Biacore T200 (GE Healthcare), the antibody SMB0002 obtained by theB cell cloning method was analyzed for its interaction with adenosine inantigen-antibody reaction. Sensor chip CM5 (GE Healthcare) wasimmobilized with an appropriate amount of protein A (Invitrogen) byamine coupling. Antibodies of interest were captured by the chip toallow interaction to adenosine as an antigen. The running buffer usedwas 50 mmol/l TrisHCl, 150 mmol/l NaCl, 0.02% (w/v) Tween20, pH 7.6. Allmeasurements were carried out at 25° C. The antigens were diluted usingthe running buffer.

Regarding SMB0002, the diluted antigen solutions and the running bufferwhich is the blank were loaded at a flow rate of 30 μL/min for 75seconds to allow each of the antigens to interact with the antibodycaptured on the sensor chip. Then, running buffer was run at a flow rateof 30 μL/min for four minutes and dissociation of the antigen from theantibody was observed. Next, 10 mmol/L glycine-HCl, pH 1.5, was loadedat a flow rate of 30 μL/min for 30 seconds to regenerate the sensorchip. Kinetic parameters such as the association rate constant ka (l/Ms)and the dissociation rate constant kd (l/s) were calculated based on thesensorgrams obtained by the measurements. The dissociation constant KD(M) was calculated from these constants. Each parameter was calculatedusing the Biacore T200 Evaluation Software (GE Healthcare).

The result showed that SMB0002 bound to adenosine. The clone wasassessed for its binding at adenosine concentrations of 100 (duplicate),50, 25, 12.5, 6.25, and 3.13 nM. The observed sensorgrams are summarizedin FIG. 8A. KD of SMB0002 toward adenosine was 1.5×10⁻⁸ (mol/L).

(3-2-2) Assessment of 2′-Adenosine-PEG-Biotin-Binding Clones for ATPBinding by the SPR Method

Biacore T200 (GE Healthcare) was used to analyze interaction in theantigen-antibody reaction with ATP. The antibody of interest wascaptured onto protein A/G (Invitrogen) immobilized in an appropriateamount onto Sensor chip CM5 (GE Healthcare) by the amine couplingmethod, and ATP which is the antigen was allowed to interact. Therunning buffer used was 10 mmol/L ACES, 150 mmol/L NaCl, 0.05% (w/v)Tween 20, pH 7.4. All measurements were carried out at 25° C. Theantigen was diluted with running buffer.

Regarding SMB0002, the diluted antigen solutions and the running bufferwhich is the blank were loaded at a flow rate of 20 μL/min for twominutes to allow each of the antigens to interact with the antibodycaptured on the sensor chip. Then, running buffer was run at a flow rateof 20 μL/min for three minutes and dissociation of the antigen from theantibody was observed. Next, 10 mmol/L glycine-HCl, pH 1.5, was injectedat a flow rate of 30 μL/min for 30 seconds to regenerate the sensorchip. Kinetic parameters such as the association rate constant ka (l/Ms)and the dissociation rate constant kd (l/s) were calculated based on thesensorgrams obtained by the measurements. The dissociation constant KD(M) was calculated from these constants. Each parameter was calculatedusing the Biacore T200 Evaluation Software (GE Healthcare).

The result showed that SMB0002 also bound to ATP. Each clone wasassessed for its binding at ATP concentrations of 5000, 1250, 313, and78.1 nM. The observed sensorgrams are summarized in FIG. 8B. As shown inFIGS. 8A and 8B, SMB0002 bound to both adenosine and ATP. The KD ofSMB0002 toward adenosine was 1.5 E⁻⁸ (mol/L) and the KD of SMB0002toward ATP was 1.0 E⁻⁵ (mol/L).

(3-2-3) Assessment of 2′-Adenosine-PEG-Biotin-Binding Clones for theirBinding to Adenosine and ATP by Competitive ELISA

Antibodies demonstrated to bind to 2′-Adenosine-PEG-Biotin were dilutedto 1 μg/ml with PBS, and added to each well of a 384-well MAXISorp(Nunc). To immobilize the antibodies, the plate was allowed to stand forone hour or more at room temperature. After the antibodies diluted withPBS were removed from each well, TBS containing 1% BSA was added theretoand the plate was allowed to stand for one hour or more. Then, the TBS(pH 7.4) containing 1% BSA was removed from the plate.2′-Adenosine-PEG-Biotin diluted to 50 nM with PBS, a mixture of2′-Adenosine-PEG-Biotin and adenosine diluted to 50 nM and 500 μMrespectively with PBS, a mixture of 2′-Adenosine-PEG-Biotin and ATPdiluted to 50 nM and 500 μM respectively with PBS, or PBS alone wasadded to the plate. The plate was allowed to stand at room temperaturefor one hour, and then washed three times with 80 μl of PBS containing0.05% Tween-20. Then, Streptavidin-HRP (Thermo fisher scientific)diluted 20000 times with PBS was added to each well, and the plate wasallowed to stand for one hour or more at room temperature. After theplate was washed three times with 80 μl of PBS containing 0.05%Tween-20, a chromogenic substrate (ABTS peroxidase substrate) was addedto each well. After the plate was incubated for one hour, colordevelopment in the solution of each well was assessed by measuringabsorbance at 405 nm using SpectraMax from Molecular Device.

As shown in FIG. 9, the result showed that the binding of SMB0002 to2′-Adenosine-PEG-Biotin was inhibited by adding excess amounts ofadenosine and ATP. Thus, the antibody clones were demonstrated to bindnot only to 2′-Adenosine-PEG-Biotin but also to both adenosine and ATP.

(3-2-4) Sequence Analysis of the Adenosine- and ATP-Binding Clone

The amino acid sequence of clone SMB0002, which bound to both adenosineand ATP, is shown in Table 3.

TABLE 3 Clone name Heavy chain SEQ ID NO Light chain SEQ ID NO SMB0002SEQ ID NO: 30 SEQ ID NO: 31

Example 4 Design of Library for Obtaining AMP/ADP/ATP/Adenosine-SwitchAntibodies Based on Comprehensive Alteration Using an Anti-ATP/AdenosineAntibody

Adenosine and ATP are known to be present at high concentrations incancer tissues and inflamed tissues. Many antibodies showingantigen-binding ability only in the presence of ATP were obtained from arational design library constructed using an ATP-binding antibody as atemplate in Reference Example 2 described below. This suggested thatantibodies showing antigen-binding ability only in the presence ofadenosine, AMP, ADP, or ATP could similarly be obtained by constructinga library using an antibody that shows binding ability to adenosine,AMP, ADP, or ATP as a template.

(4-1) Assessment of the Binding of the Adenosine-Binding AntibodySMB0002 to AMP and ADP Binding by the SPR Method

SMB0002 expressed and purified by the method described in ReferenceExample 1 described below was assayed for AMP binding by a methodsimilar to the measurement method using Biacore described in Example3-2. Binding of SMB0002 was assessed at AMP concentrations of 500, 250(duplicate), 125, 62.5, 31.25, 15.625, and 7.8125 μM. The observedsensorgrams are shown in FIG. 10A. As shown in FIG. 10A, binding ofSMB0002 to AMP was observed. The KD of SMB0002 toward AMP was 5.9×10⁻⁵(mol/L).

Binding to ADP was assessed by a method similar to the measurementmethod using Biacore described in Example 3-2, except that the NaClconcentration was changed to 600 mM. Binding of SMB0002 was assessed atADP concentrations of 2000, 1000 (duplicate), 500, 250, 250, 125, 62.5,and 31.3 μM. The observed sensorgrams are shown in FIG. 10B. As shown inFIG. 10B, binding of SMB0002 to ADP was observed. The KD of SMB0002toward ADP was 2.4×10⁻⁴ (mol/L).

(4-2) X-Ray Crystallographic Analysis of the Adenosine-Binding AntibodySMB0002

The three-dimensional structure of the complex of adenosine and theadenosine-binding antibody SMB0002 obtained from immunized rabbits inExample 3 was revealed by X-ray crystallographic analysis.

(4-2-1) Preparation of Full-Length SMB0002 Antibody for Crystallization

The full-length SMB0002 antibody for crystallization was prepared andpurified by a method known to those skilled in the art.

(4-2-2) Preparation of SMB0002 Fab Fragments from the Full-LengthAntibody

After the obtained full-length SMB0002 antibody was concentrated with a10000 molecular weight cutoff (MWCO) ultrafiltration membrane, a samplewas prepared by diluting to 1.5 mg/ml with 4 mM L-cysteine, 5 mM EDTA,25 mM MES, pH 6.5. Papain (Roche Applied Science) was added to thesample at an amount of 1/100 to the full-length antibody by mass ratio,and this was allowed to stand at 35° C. for 2 hours. Then, the reactionwas terminated by adding 20 ml of 25 mM sodium acetate buffer, pH 5.0,in which a tablet of protease inhibitor cocktail mini, EDTA-free (RocheApplied Science) was dissolved. Next, this sample was loaded onto a 1-mlsize cation-exchange column HiTrap SP HP (GE Healthcare) to whosedownstream 1-ml size Protein A-carrying column HiTrap MabSelect Sure (GEHealthcare) was tandemly connected and which was equilibrated with 25 mMsodium acetate buffer, pH 5.0. Elution was performed by linearlyincreasing the concentration of NaCl in the buffer, and a purifiedfraction of Fab fragments of the SMB0002 antibody was obtained. Then,the obtained purified fraction was concentrated with a 5000 MWCOultrafiltration membrane and loaded onto the gel filtration columnSuperdex 200 16/60 prep grade (GE Healthcare) equilibrated with 25 m MHEPES buffer, pH 7.0, 100 mM NaCl. The column was eluted with the samebuffer to obtain Fab fragments of SMB0002 for crystallization. Allcolumn operations were carried out at low temperature.

(4-2-3) Crystallization of the Complex of Adenosine and SMB0002 FabFragment

A sample of SMB0002_Fab for crystallization purified by theabove-described method was concentrated with a 5000 MWCO ultrafiltrationmembrane to A280=22.3. Then, adenosine was added at a finalconcentration of 0.9 mM, and crystallization was carried out using thesitting drop vapor diffusion method. Using a reservoir solution of 20%PEG3350 and 0.2M ammonium citrate dibasic, crystallization drops wereprepared by mixing at reservoir solution: crystallization sample=0.2μl:0.2 μl by Hydra II Plus One (MATRIX). The drops were allowed to standat 20° C. and plate-like crystals were successfully obtained.

(4-2-4) Measurement of X-Ray Diffraction Data from the Crystal of theComplex of SMB0002 Fab Fragment and Adenosine

An obtained single crystal of the complex of SMB0002 Fab fragment andadenosine was immersed in a solution of 0.2 M ammonium citrate dibasic,0.025 M HEPES pH 7, 25% PEG3350, 0.1 M NaCl, 1 mM Adenosine, and 16%Glycerol. Then, the single crystal was scooped together with thesolution using a pin equipped with a minute nylon loop, and frozen inliquid nitrogen. X-ray diffraction data were measured at BL-17A of thesynchrotron radiation facility Photon Factory of the High EnergyAccelerator Research Organization. The frozen state was maintainedthrough the measurement by placing in a stream of nitrogen gas at −178°C. A total of 300 X ray diffraction images were collected using the CCDdetector Quantum 315r (ADSC) attached to the beamline by rotating thecrystal by 0.6°. Lattice constant determination, diffraction spotindexing, and diffraction data processing from the obtained diffractionimages were performed using the programs Xia2 (J. Appl. Cryst. (2010)43, 186-190), XDS Package (Acta Cryst. (2010) D66, 125-132), and Scala(Acta Cryst. (2006) D62, 72-82). Ultimately, this successfully yieldeddiffraction intensity data of up to 1.76 angstrom resolution. Thiscrystal belonged to space group P1 with lattice constants a=49.960angstrom, b=105.730 angstrom, c=106.166 angstrom, α=62.58°, β=77.29°,γ=77.49°.

(4-2-5) X-Ray Crystallographic Analysis of the Complex of Adenosine andSMB0002 Fab Fragment

To determine the structure of the SMB0002 Fab fragment and adenosinecomplex crystal, the molecular replacement method was carried out usingthe program Phaser (J. Appl. Cryst. (2007) 40, 658-674). The number ofcomplexes in the asymmetrical unit was estimated to be four from thesize of the obtained crystal lattice and the molecular weight of theSMB0002 Fab fragment. A homology model of the antibody was constructedusing Discovery Studio 3.5 (Accelrys). The model was divided into thevariable region and constant region, and using the coordinate of eachstructure as search model, their orientation and position in the crystallattices were determined based on the rotation function and translationfunction. Further, the crystallographic reliability factor R for thediffraction intensity data at 25 to 3.0 angstroms was 46.36% and Free Rwas 46.10%, when rigid body refinement was carried out on the obtainedinitial structural model in which the variable region and constantregion portions were independently moved. Then, structural modelrefinement was carried out by repeating the following processes:structural refinement using the program REFMAC5 (Acta Cryst. (2011) D67,355-367), and revision of the structural model performed using theprogram Coot (Acta Cryst. (2010) D66, 486-501) by referring to theelectron density maps having as coefficients 2Fo-Fc and Fo-Fc, whichwere calculated based on the experimentally determined structural factorFo, the structural factor Fc which were calculated from the model, andthe phase calculated from the model. Ultimately, with 168160 diffractionintensity data at 25 to 1.76 angstrom resolution, the crystallographicreliability factor R and Free R of the structural model containing 14681non-hydrogen atoms were 19.82% and 23.15% respectively.

(4-2-6) Identification of the Interaction Sites of SMB0002 and Adenosine

Ultimately, the crystallographic structure of the complex of SMB0002_Fabfragment and adenosine was determined at a resolution of 1.76 angstrom.There were four SMB0002_Fab fragments in the asymmetrical unit of thecrystal, adenosine was bound to all of them, and the binding mode wasalmost the same for all. The crystallographic structure showed thatadenosine bound in a pocket formed between the H chain and L chain ofthe Fab fragment of the antibody, in a manner that the adenine ring isoriented toward the depth of the pocket. As shown in FIG. 11A, theadenine ring moiety of adenosine is recognized by each of the sidechains of H-chain A33, I50, W58, and Y100 and L-chain Y95c and N96, aswell as by each of the main chains of H-chain G99 and T100a of theantibody. It was revealed that robust recognition was achieved inparticular by formation of two hydrogen bonds between the side chain ofL-chain N96 and both N at position 1 and NH2 at position 6 of adenosine,as well as by formation of hydrogen bonds between the main chaincarbonyl oxygen and amide NH group of H-chain T100a and NH2 at position6 and N at position 7 of the adenine ring respectively. Furthermore, theadenine ring is surrounded by each of the side chains of H-chain A33,ISO, W58, Y100 and L-chain Y95c of the antibody, and forms van der Waalsinteractions and CH-π interactions with these residues. Both H-chain G99and T100a form an interaction with the adenine ring in their main chainmoieties. However, since G99 has a φ-Ψ angle characteristic of Gly onthe Ramachandran plot, it is thought to be important in maintaining theloop structure of the H-chain CDR3 upon binding to adenosine. Moreover,the side chain of T100a is also thought to play an important role inmaintaining the loop structure of the H-chain CDR3 upon binding toadenosine by forming an interaction with other residues in the H-chainCDR3. As shown in FIG. 11B, the ribose moiety of adenosine is recognizedby the respective side chains of H-chain S56 and W58 and L-chain Y95c,as well as the T57 main chain and H-chain G52. Interaction with theseresidues is primarily attributed to van der Waals interaction; however,formation of a hydrogen bond is seen, albeit weak, between the sidechain of H-chain S56 and the 3′ OH of ribose. H-chain G52, including itsCa atom, forms multiple van der Waals interactions with the ribosemoiety, and is thought to play an important role in adenosinerecognition. Meanwhile, H-chain T57 forms an interaction with the ribosemoiety at its main chain only, and the side chain is not directlyinvolved in the binding.

As shown in Example 4-1, the antibody binds not only to adenosine butalso to AMP, though with reduced binding activity. In thecrystallographic structure, the 5′ OH of ribose in adenosine is formingan intramolecular interaction with N at position 3 of the adenine ringmoiety; however, this binding cannot be formed in AMP and the positionof the 5′ O slightly changes so that, as a result, the phosphate groupof AMP is inferred to be present in the region indicated by the dottedline in FIG. 11B. Since this region is in a position that allowsinteraction with residues in the H-chain CDR2 and L-chain CDR3, bindingto AMP can be expected to be increased by introducing appropriatemutations into the H-chain CDR2 and L-chain CDR3.

From the results described above, the mode of adenosine recognition bythe antibody has been revealed and the amino acid residues of theantibody variable region that are greatly involved in adenosine bindinghave been identified. The amino acid residues that are greatly involvedin adenosine binding include: A33, I50, G52, S56, T57, W58, G99, Y100,and T100a (Kabat numbering) in the H chain, and Y95c and N96 (Kabatnumbering) in the L chain. Moreover, predicted residues that arepossibly located close to the 5′ phosphate group in AMP were: D54, S55,S56, T57, and W58 of the H-chain CDR2, and G95a, W95b, and Y95c of theL-chain CDR3. Modification of these residues may result in increasedbinding with AMP.

Furthermore, by performing similar considerations for ADP and ATP basedon crystallographic structures, modifications that can increase thebinding to ADP and ATP could also be predicted.

(4-3) Humanization of Rabbit-Derived Antibody SMB0002

SMB0002 is a rabbit-derived antibody; thus, to construct a humanantibody library, the sequence was humanized by a method known to thoseskilled in the art (EP Patent Publication No. 239400; InternationalPublication Nos. WO1996/002576; WO1993/012227; WO1992/003918;WO1994/002602; WO1994/025585; WO1996/034096; WO1996/033735;WO1992/001047; WO1992/020791; WO1993/006213; WO1993/011236;WO1993/019172; WO1995/001438; WO1995/015388; Cancer Res., (1993) 53,851-856; BBRC., (2013) 436 (3):543-50; etc.).

Humanized SMB0002 (heavy chain variable region sequence: SEQ ID NO: 85;light chain variable region sequence: SEQ ID NO: 86) was expressed andpurified by the method described in Reference Example 1-1. Binding ofhumanized SMB0002 to adenosine and AMP was measured and analyzed by amethod using Biacore T200 (GE Healthcare). Protein A (Invitrogen)immobilized in an appropriate amount onto a Sensor chip CM5 (GEHealthcare) by the amine coupling method was allowed to capture theantibody of interest, and the interaction with the antigen adenosine,AMP, ADP, or ATP was observed. The running buffer used was 50 mMTris-HCl, 150 mM NaCl, 0.02% (w/v) Tween 20, pH 7.6, for adenosine andAMP, and 50 mM Tris-HCl, 150 mM NaCl, 0.02% (w/v) Tween 20, 2 mM MgCl₂,pH 7.6, for ADP and ATP. All measurements were carried out at 25° C. Theantigens were diluted with the running buffer.

Diluted antigen solutions and the running buffer as a blank were addedat a flow rate of 30 μL/min for 75 seconds to the antibody captured onthe sensor chip, and the binding between the antibody and antigens wasobserved. Then, the running buffer was run at a flow rate of 30 μL/minfor five minutes, and dissociation of the antigens from the antibody wasobserved. Next, 10 mM glycine-HCl, pH 1.5, was added at a flow rate of30 μL/min for 30 seconds to regenerate the sensor chip. Kineticparameters such as the association rate constant ka (l/Ms) and thedissociation rate constant kd (l/s) were calculated based on thesensorgrams obtained by the measurements. The dissociation constant KD(M) was calculated from these constants. Each parameter was calculatedusing Biacore T200 Evaluation Software (GE Healthcare).

The result showed that humanized SMB0002 bound to adenosine, AMP, ADP,and ATP. Sensorgrams observed when samples with adenosine concentrationsof 200, 100, 50 (duplicate), 25, 12.5, 6.25, and 3.125 nM interactedwith the clone are summarized in FIG. 12. The KD of humanized SMB0002toward adenosine was 7.5×10⁻⁹ M. Next, sensorgrams observed when sampleswith AMP concentrations of 500, 250, 125 (duplicate), 62.5, 31.3, 15.6,and 7.8 μM interacted with the clone are summarized in FIG. 13. The KDof humanized SMB0002 toward AMP was 3.5×10⁻⁵ M. Next, sensorgramsobserved when samples with ADP concentrations of 1000 (duplicate), 500,250, 125, and 62.5 μM interacted with the clone are summarized in FIG.14. The KD of humanized SMB0002 toward ADP was 7.9×10⁻⁵M. Finally,sensorgrams observed when samples with ATP concentrations of 1000(duplicate), 500, 250, 125, and 62.5 μM interacted with the clone aresummarized in FIG. 15. The KD of humanized SMB0002 toward ATP was1.4×10⁻⁴ M. Since humanized SMB0002 had binding activity towardadenosine, AMP, ADP, and ATP, the sequence was used as a templatesequence for constructing a human antibody library.

(4-4) Assessment of Comprehensive Variants for Designing a Library Basedon the Result of X-Ray Crystallographic Structure Analysis

The crystallographic structure of the complex of adenosine and theadenosine-binding antibody SMB0002 was analyzed in Example (4-2). Themode of recognition by which the antibody recognizes adenosine (and AMP)and the amino acid residues of the antibody variable region that arepresumed not to be significantly involved in adenosine (and AMP) bindingwere deduced based on the result of crystallographic structure analysis.It was conceived that, by comprehensively evaluating variants whoseresidues located close to the adenosine recognition site are substitutedwith each of the amino acids, the sites that can be made into a libraryand the amino acids that can be made into a library could be determined.Specifically, it was conceived that, by evaluating the sites that arenot greatly involved in the binding toward adenosine, AMP, ADP, or ATP,the sites in which amino acids other than those of the native sequencethat may be involved in binding but do not significantly reduce thebinding toward adenosine, AMP, ADP, or ATP (do not render the binding tozero) are present, as well as the amino acids, the sites that can bemade into a library and the amino acids that can be made into a librarycould be determined. Several variants were generated by introducingmodifications to these residues in the humanized SMB0002 prepared inExample (4-3).

Of the sites in the heavy chain, the modified sites (the sites shownaccording to Kabat numbering and indicated as “Kabat” in the table), theamino acids before modification at these sites (the amino acidsindicated as “native sequence” in the table), and the amino acids aftermodification (the amino acids indicated as “altered amino acids” in thetable) are shown in Table 4.

TABLE 4 HCDR1 HCDR2 HCDR3 Kabat 31 32 53 54 55 56 57 58 61 62 65 96 9798 99 100 100a 100b 100c 100d 101 102 Native sequence N Y A D S S T Y SW G R F V G Y T N A F D P Altered A A A A A A A A A A A A A A A A A A AA amino D D D D D D D D D D D D D D D D D acid E E E E E E E E E E E E EE E E E E F F F F F F F F F F F F F F F F F F G G G G G G G G G G G G GG G G G G G G H H H H H H H H H H H H H H H H H I I I I I I I I I I I II I I I I K K K K K K K K K K K K K K K K K K L L L L L L L L L L L L LL L L L L N N N N N N N N N N N N P P P P P P P P P P P P P P P P Q Q QQ Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q R R R R R R R R R R R R R R R R R S S SS S S S S S S S S S S S S T T T T T T T T T T T T T T T T T T V V V V VV V V V V V V V V V V V V W W W W W W W W W W W W W W W W W W Y Y Y Y YY Y Y Y Y Y Y Y Y

Of the sites in the light chain, the modified sites (the sites shownaccording to Kabat numbering and indicated as “Kabat” in the table), theamino acids before modification at these sites (the amino acidsindicated as “native sequence” in the table), and the amino acids aftermodification (the amino acids indicated as “altered amino acids” in thetable) are shown in Table 5.

TABLE 5 LCDR1 LCDR3 Kabat 28 29 32 93 94 95 95a 95b 95c Native sequenceW N Y A N S G W Y Altered A A A A A A A A A amino D D D D D D D D D Dacid E E E E E E E E E E F F F F F F F F F F G G G G G G G G G H H H H HH H H H H I I I I I I I I I I K K K K K K K K K K L L L L L L L L L L NN N N N N P P P P P P P P P P Q Q Q Q Q Q Q Q Q Q R R R R R R R R R R SS S S S S S S T T T T T T T T T V V V V V V V V V V W W W W W W W W Y YY Y Y Y Y Y

The binding of each variant expressed and purified by the methoddescribed in Reference Example 1 described below to adenosine and AMPwas measured by the method using Biacore described in Example 4-3 exceptthat the MgCl₂ concentration was 2 mM. As a result of the measurements,the affinity of each variant toward adenosine and AMP was calculated asKD value. The result of the comparison of the KD values for adenosinebetween each variant of the heavy chain and the parental sequence,humanized SMB0002, is shown in Table 6, and the result of the comparisonof the KD values for AMP is shown in Table 7. The result of thecomparison of the KD values for adenosine of each variant of the lightchain and humanized SMB0002 is shown in Table 8, and the result of thecomparison of the KD values for AMP is shown in Table 9.

TABLE 6 HCDR1 HCDR2 HCDR3 Kabat 31 32 53 54 55 56 57 59 61 62 65 96 9798 99 100 100a 100b 100c 100d 101 102 Native sequence N Y A D S S T Y SW G R F V G Y T N A F D P Altered A 0.9 0.1 0.0 0.5 0.4 0.3 0.9 1.0 0.70.8 0.5 0.3 0.7 0.4 0.0 0.2 0.1 0.7 0.2 amino D 0.6 0.1 0.5 0.5 0.4 0.20.7 0.0 1.0 0.7 0.2 0.3 0.4 0.4 0.0 0.3 acid E 0.7 0.1 0.5 0.3 0.5 0.40.2 0.8 0.9 0.7 1.0 0.3 0.5 0.2 0.0 0.6 0.2 F 0.7 0.7 0.2 0.5 0.5 0.20.2 0.6 1.1 0.8 0.9 1.6 0.6 0.1 0.9 0.5 0.1 G 0.5 0.3 0.8 0.3 0.6 0.30.2 0.7 0.9 0.7 0.2 0.4 0.0 0.0 0.1 0.1 0.0 0.6 0.2 H 0.4 0.7 0.2 0.30.6 0.3 0.2 0.9 0.6 0.7 0.5 0.6 0.2 0.2 0.6 0.1 I 1.1 0.3 0.4 0.2 0.40.2 0.2 0.7 0.7 0.2 0.4 0.9 0.1 0.0 0.5 0.2 K 0.9 0.5 0.8 0.1 0.3 0.40.2 0.6 0.5 0.7 0.9 0.4 0.7 0.1 0.0 0.6 0.2 L 0.9 0.2 0.2 0.4 0.3 0.60.2 0.6 0.6 0.7 0.6 0.3 0.7 0.1 0.0 0.5 0.1 N 0.5 0.3 0.2 0.8 0.7 0.40.4 0.6 0.0 0.5 0.3 P 0.3 1.6 0.3 0.1 0.2 0.1 0.2 0.1 0.8 0.0 0.1 0.80.1 0.0 0.0 Q 1.2 0.3 0.5 0.4 0.3 0.3 0.5 0.7 1.1 0.8 0.6 0.4 0.8 0.10.0 0.1 0.5 0.2 R 0.8 0.8 0.5 0.7 0.4 0.5 0.3 0.6 0.9 0.8 0.3 0.9 0.10.0 0.4 0.2 S 1.0 0.2 0.3 0.4 0.3 0.9 0.6 0.3 0.9 0.3 0.0 0.2 0.1 0.70.2 T 1.0 0.3 0.4 0.5 0.3 0.5 0.6 1.1 0.6 0.9 0.3 0.6 0.1 0.0 0.1 0.40.1 V 0.6 0.2 0.4 0.3 0.3 0.2 0.5 0.6 1.0 0.6 0.4 0.4 0.0 0.0 0.5 0.40.1 W 0.6 0.4 0.0 0.2 0.1 0.1 0.2 0.5 0.9 0.7 0.9 0.5 0.5 0.0 0.1 0.50.1 Y 0.7 0.2 0.2 0.3 0.2 0.2 1.0 0.9 0.4 0.5 0.1 0.6 0.1

TABLE 7 HCDR1 HCDR2 HCDR3 Kabat 31 32 53 54 55 56 57 59 61 62 65 96 9798 99 100 100a 100b 100c 100d 101 102 Native sequence N Y A D S S T Y SW G R F V G Y T N A F D P Altered A 0.9 0.3 0.0 0.9 1.1 0.4 1.0 1.3 0.90.9 0.6 0.3 0.7 0.1 0.0 0.1 0.0 2.5 0.2 amino D 0.6 0.2 2.1 0.8 0.9 0.30.7 0.0 1.0 1.0 0.2 0.3 0.4 0.1 0.0 0.5 acid E 1.1 0.2 1.6 1.2 0.6 0.80.2 1.0 0.9 1.1 1.0 0.3 0.6 0.2 0.0 1.0 0.2 F 1.1 1.1 0.3 0.8 0.6 0.20.2 0.9 1.3 1.0 1.1 2.0 0.7 0.1 1.3 0.8 0.2 G 0.6 0.3 2.0 1.0 0.8 0.60.3 0.8 1.2 0.8 0.3 0.4 0.0 0.0 0.0 0.1 0.0 0.7 0.3 H 1.1 0.9 0.2 1.11.0 0.6 0.2 1.1 1.3 0.8 0.9 0.8 0.1 0.1 1.1 0.2 I 1.3 0.5 0.7 0.3 0.40.3 0.3 0.7 0.8 0.3 0.5 1.0 0.0 0.0 0.8 0.2 K 1.0 1.4 2.7 0.0 0.6 0.90.4 0.8 1.2 0.9 1.3 0.6 0.8 0.1 0.0 0.9 0.3 L 1.1 0.3 0.5 0.7 0.6 0.90.4 0.7 1.2 0.9 0.8 0.3 0.9 0.1 0.0 0.7 0.2 N 0.8 0.7 0.3 1.6 0.8 0.50.4 0.7 0.0 0.8 0.4 P 0.3 3.2 0.6 0.3 0.2 0.1 0.2 0.2 1.2 0.0 0.1 0.80.1 0.0 0.0 Q 1.2 0.3 1.2 0.8 0.5 0.6 0.6 0.9 1.6 0.7 0.7 0.4 0.9 0.10.0 0.0 0.7 0.3 R 1.2 1.4 1.2 0.0 0.6 1.2 0.4 0.9 1.2 0.8 0.4 1.0 0.10.0 0.7 0.3 S 1.3 0.3 0.7 1.0 0.4 1.0 1.0 0.6 0.8 0.1 0.0 0.2 0.0 0.80.3 T 1.6 0.4 0.9 1.5 0.7 1.1 1.0 1.2 0.9 1.3 0.5 0.8 0.0 0.0 0.0 0.60.2 V 1.5 0.6 0.6 0.4 0.4 0.3 0.6 0.8 1.3 0.8 0.6 0.3 0.0 0.0 0.5 0.50.2 W 1.4 1.2 0.0 0.4 0.0 0.1 0.2 1.1 1.1 0.7 1.1 0.5 0.1 0.0 0.0 0.80.2 Y 1.3 0.4 0.4 0.3 0.2 0.2 1.1 1.0 0.5 0.1 0.0 0.9 0.2

TABLE 8 LCDR1 LCDR3 Kabat 28 29 32 93 94 95 95a 95b 95c Native sequenceW N Y A N S G W Y Altered A 0.3 0.7 0.2 0.9 0.7 0.5 0.7 0.1 amino D 0.10.7 0.1 0.4 1.0 0.6 0.5 0.4 0.1 acid E 0.1 0.7 0.2 0.4 0.7 0.7 0.5 0.60.1 F 0.8 0.4 0.7 0.6 0.7 0.4 0.4 0.5 0.6 G 0.1 0.7 0.2 0.3 1.1 0.9 0.70.1 H 0.6 0.6 0.4 0.8 0.7 0.8 1.0 0.8 0.3 I 0.1 0.4 0.0 0.1 0.6 0.8 0.50.7 0.2 K 0.3 0.6 0.1 0.0 0.8 0.7 0.5 0.5 0.5 L 0.2 0.6 0.1 0.7 0.6 0.80.6 0.5 1.0 N 0.3 0.5 1.0 0.5 0.6 P 0.1 0.3 0.1 0.1 0.3 0.6 0.3 0.4 0.1Q 0.1 0.8 0.1 0.8 0.6 0.6 0.7 0.8 0.0 R 0.2 0.7 0.1 1.3 0.6 0.6 0.8 1.10.2 S 0.6 0.7 0.2 0.5 0.9 0.8 0.1 T 0.5 0.4 0.1 0.7 0.7 0.7 0.9 0.1 V0.6 0.4 0.0 0.5 0.4 0.7 0.6 0.7 0.2 W 0.6 0.1 0.0 0.7 0.5 0.5 0.1 Y 0.70.7 0.8 0.7 0.3 0.3 1.0

TABLE 9 LCDR1 LCDR3 Kabat 28 29 32 93 94 95 95a 95b 95c Native sequenceW N Y A N S G W Y Altered A 0.4 0.8 0.2 1.3 1.4 0.8 1.2 0.2 amino D 0.11.0 0.1 0.8 1.2 1.2 0.8 1.0 0.1 acid E 0.1 1.0 0.2 0.8 1.2 0.9 1.0 1.20.0 F 0.9 0.6 0.9 1.0 1.3 0.3 1.1 0.9 1.1 G 0.1 1.2 0.2 0.5 1.5 1.8 1.00.1 H 0.9 0.9 0.5 1.2 1.1 1.0 0.8 1.0 0.7 I 0.2 0.6 0.1 0.1 0.9 0.8 0.50.9 0.3 K 0.4 1.0 0.1 0.0 1.1 0.7 0.6 1.5 2.2 L 0.3 1.2 0.1 0.9 1.3 1.81.4 1.3 1.9 N 0.4 1.0 0.7 1.1 1.9 P 0.1 0.5 0.1 0.0 0.5 1.4 0.4 0.8 0.3Q 0.1 1.0 0.1 1.3 1.5 1.2 1.0 1.4 0.0 R 0.2 1.0 0.1 2.5 1.0 0.9 1.0 2.20.4 S 0.5 1.0 0.3 1.0 1.6 1.6 0.1 T 0.5 0.6 0.1 1.2 1.0 1.1 1.4 0.1 V0.5 0.8 0.0 0.8 0.6 1.0 0.5 1.2 0.4 W 1.0 0.2 0.0 0.8 0.7 1.0 0.1 Y 1.00.9 1.3 1.4 0.5 0.4 0.9

(4-5) Library Design Based on Comprehensive Variant Evaluation

To design a library, sites that meet at least one of the conditionsshown below were selected as library-constructible sites based on theinformation obtained in Example 4-4.

Condition 1: sites that are not greatly involved in the binding towardadenosine, AMP, ADP, or ATP, or sites in which amino acids other thanthose of the native sequence that may be involved in binding but do notsignificantly reduce the binding toward adenosine, AMP, ADP, or ATP (donot render the binding to zero) are present;

Condition 2: sites having a certain level of diversity of amino acidoccurrence frequency as repertoire of the antibody; and

Condition 3: sites that are not important for the formation of canonicalstructures.

From the evaluation results of Example (4-4), sites for which at leastone or more variants exist, which variants have KD values towardadenosine and AMP both indicating more than 20% binding of the parentsequence (humanized SMB0002) toward adenosine and AMP, were judged to bemodifiable sites that meet the above-described conditions. Of the aminoacids substituted at these sites, the amino acids whose KD values towardadenosine and AMP both indicate more than 20% binding of the parentsequence (humanized SMB0002) toward adenosine and AMP were judged to belibrary-constructible amino acids (flexible residues that can be made toappear in the library). A library for obtaining ATP/AMP/ADP/adenosineswitch antibodies is constructed by designing a library in which atleast any one or more amino acids from among the amino acids containedin the amino acid repertoire, which includes the library-constructibleamino acids selected from the above-described variant analysis (theflexible residues that can be made to appear in the library) and theamino acids of the non-modified antibody (i.e., the amino acids includedin the native sequence of the humanized SMB0002), appear at thedetermined modifiable sites in the CDRs of the humanized SMB0002. Thesites containing an amino acid repertoire in the heavy chain, and theamino acid repertoires at these sites are shown in Table 10. The sitescontaining an amino acid repertoire in the light chain, and the aminoacid repertoires at these sites are shown in Table 11. In the tables,sites shown according to Kabat numbering indicated by “Kabat” representmodifiable sites; amino acids indicated by “native sequence” representnon-modified amino acids at these sites; and amino acids indicated by“library-constructible amino acids” represent library-constructibleamino acids at these sites. A library, in which at least any one aminoacid from among the amino acids contained in the selected amino acidrepertoire appear at each of the modifiable sites, was designed.

TABLE 10 HCDR1 HCDR2 HCDR3 Kabat 31 32 53 54 55 56 57 59 61 62 65 96 9798 100 100a 101 102 Native sequence N Y A D S S T Y S W G R F V Y T D PLibrary- A A A A A A A A A A A A A construct- D D D D D D D D D D D Dible amino E E E E E E E E E E E E E acid F F F F F F F F F F F F F F FG G G G G G G G G G G G G H H H H H H H H H H H H I I I I I I I I I I II I K K K K K K K K K K K K K K L L L L L L L L L L L L L L N N N N N NN N N N N P P P P P P Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q R R R R R R R R R RR R R S S S S S S S S S S S T T T T T T T T T T T T T T V V V V V V V VV V V V V V V W W W W W W W W W Y Y Y Y Y Y Y Y Y

TABLE 11 LCDR1 LCDR3 Kabat 28 29 32 93 94 95 95a 95b 95c Native sequenceW N Y A N S G W Y Library- A A A A A A A A construct- D D D D D D D Dible amino E E E E E E E acid F F F F F F F F F F G G G G G G G H H H HH H H H H H I I I I I I K K K K K K K K L L L L L L L L N N N N N N P PP P P P Q Q Q Q Q Q Q R R R R R R R S S S S S S T T T T T T T V V V V VV V V V W W W W W Y Y Y Y Y Y Y Y

Genes containing each of the sequences included in the library designedin this manner are synthesized, and using the collection (library) ofthese individual genes as a template, a gene library is amplified withprimers that can amplify each of VH and VL. The amplified gene libraryof rationally designed human antibody heavy chain variable regions andthat of the human antibody light chain variable regions are introducedinto appropriate phagemid vectors having both a human IgG-derived CH1sequence and a human IgG-derived light chain constant region sequence. Arationally designed library which enables to obtain antibodies whichbind to antigens using adenosine, AMP, ADP or ATP as a switch isconstructed by introducing these phagemid vectors into Escherichia coliby electroporation and then presenting Fab domains consisting of a humanantibody variable region-constant region. Such a rationally designedlibrary composed of diverse H chains and L chains having bindingactivities to adenosine, AMP, ADP, or ATP is thought to be useful as alibrary containing human antibodies that enable to efficiently obtainAMP/ADP/ATP/adenosine switch antibodies against arbitrary antigens,where adenosine, AMP, ADP, or ATP is sandwiched between the antibody andantigen as shown in FIG. 43. Moreover, since, as described above,SMB0002 binds not only to adenosine and AMP but also to ADP and ATP, itis also predicted to have binding activity to cAMP and ATP-gamma-S whichare structurally similar to AMP, ADP, ATP, and adenosine. Thus, thelibrary is considered to be useful for obtaining switch antibodies whosebinding activity toward an arbitrary target antigen varies depending onthe presence or absence of any one or more small molecules of ATP, ADP,AMP, cAMP, adenosine, and ATP-gamma-S.

Example 5 Design of Library for Obtaining AMP/ADP/ATP/Adenosine SwitchAntibodies by Panning with Molecules Acting as a Switch

A method which involves designing library sites by comprehensivealteration and constructing a library using as template an antibodyshowing binding ability toward adenosine, AMP, ADP, or ATP was describedin Example 4. As a different approach, a method using panning onmolecules that act as a switch is also useful as a method for generatinga library.

(5-1) X-Ray Crystallographic Structure Analysis of the Adenosine-BindingAntibody SMB0002

The crystallographic structure of the complex of adenosine and theadenosine-binding antibody SMB0002 was revealed in Example 4-2. The modeof recognition by which the antibody recognizes adenosine (and AMP) andthe amino acid residues of the antibody variable region that arepresumed not to be significantly involved in adenosine (and AMP) bindingwere deduced based on the result of crystallographic structure analysis.

SMB0002 is a rabbit-derived antibody; thus, to construct a humanantibody library, the sequence was humanized by a method known to thoseskilled in the art (as described above).

To design a library, sites that meet at least one of the followingconditions are selected as library-constructible sites.

Condition 1: sites that are not greatly involved in the binding towardadenosine, AMP, ADP, or ATP, or sites in which amino acids other thanthose of the native sequence are present and which may be involved inbinding but which do not significantly reduce the binding towardadenosine, AMP, ADP, or ATP (do not render the binding to zero);

Condition 2: sites having a certain level of diversity of amino acidoccurrence frequency as repertoire of the antibody; and

Condition 3: sites that are not important for the formation of canonicalstructures.

First, a library is designed, in which the occurrence of nucleotides forthe amino acids at sites in the CDR1, CDR2, or CDR3 among the sitescontained in the humanized SMB0002 sequence and fulfilling the aboveconditions in the heavy chain, and which are not greatly involved in thebinding toward adenosine, AMP, ADP, or ATP, are limited to onlyparticular nucleotides. Examples include NNK and TRIM libraries(Gonzalez-Munoz A et al. MAbs 2012; Lee C V et al. J Mol Biol. 2004;Knappik A. et al. J Mol Biol. 2000; Tiller T et al. MAbs 2013). Genes ofthe collection of designed gene sequences are synthesized (heavy chainvariable region library), combined with the light chain variable regionsequence of the humanized SMB0002 (the parental sequence, SEQ ID NO:86), and introduced into appropriate phagemid vectors having a humanIgG-derived CH1 sequence and a human IgG-derived light chain constantregion sequence. These phagemid vectors are introduced into Escherichiacoli by electroporation to construct a phage-display library of humanantibody heavy chain variable regions capable of binding to antigensusing any one of adenosine, AMP, ADP, and ATP as a switch.

Then, a library is designed, in which the occurrence of nucleotides forthe amino acids at sites in the CDR1, CDR2, or CDR3 among the sitescontained in the humanized SMB0002 sequence and fulfilling the aboveconditions in the light chain, and which are not greatly involved in thebinding toward adenosine, AMP, ADP, or ATP, are limited to onlyparticular nucleotides. Examples include NNK and TRIM libraries. Genesof the collection of designed gene sequences are synthesized (lightchain variable region library), combined with the heavy chain variableregion sequence of the humanized SMB0002 (the parental sequence, SEQ IDNO: 85), and introduced into appropriate phagemid vectors having a humanIgG-derived CH1 sequence and a human IgG-derived light chain constantregion sequence. These phagemid vectors are introduced into Escherichiacoli by electroporation to construct a phage-display library of humanantibody light chain variable regions capable of binding to antigensusing any one of adenosine, AMP, ADP, and ATP as a switch.

Panning is performed using biotinylated ATP, biotinylated ADP,biotinylated AMP, or biotinylated adenosine to obtain a population ofantibodies that specifically bind to ATP, ADP, AMP, or adenosine fromeach of the constructed phage-display libraries of heavy chain and lightchain variable regions.

Biotinylated adenosine was prepared by the method described in Example2-2-11. ATP-PEG-biotin can be purchased from JenaBioscience (Catalog No.NU-926-BIO) and used. Like ATP, AMP and ADP have a structure in which aphosphate group is added to the 5′ hydroxyl group of adenosine. Thus, itis preferable that an appropriate linker (such as a PEG linker) isattached to the phosphate group and then biotin is added to the end ofthe linker in the same fashion as in biotinylated ATP.

Phages are produced by E. coli retaining the constructed phagemids forphage-display. The phage population precipitated by adding 2.5 MNaCl/10% PEG to the culture solution of E. coli producing phages isdiluted with TBS to obtain a phage library suspension. Then, BSA isadded at a final concentration of 4% to the phage library suspension.Panning is performed using antigens immobilized onto magnetic beads. Asmagnetic beads, NeutrAvidin coated beads (Sera-Mag SpeedBeadsNeutrAvidin-coated) or Streptavidin coated beads (Dynabeads M-280Streptavidin) can be used.

Phages capable of binding to adenosine, AMP, ADP, or ATP are collectedby panning. Specifically, by adding biotin-labeled antigens to theprepared phage library suspension, the phage library is contacted withadenosine, AMP, ADP, or ATP at room temperature for 60 minutes.BSA-blocked magnetic beads are added, and the antigen/phage complex isallowed to bind to the magnetic beads at room temperature for 15minutes. The beads are washed with 1 mL of TBST and TBS. Then, beads towhich TBS containing trypsin at a final concentration of 1 mg/mL hasbeen added are suspended for 15 minutes at room temperature, and a phagesuspension is collected from the beads separated immediately thereafterusing a magnetic stand. The pIII protein of phages not displaying Fab(helper phage-derived pIII protein) is cleaved by addition of trypsin,resulting in loss of the ability of phages not displaying Fab to infectE. coli. Phages eluted with the trypsin solution are added to 10 mL ofthe E. coli strain ER2738 in a logarithmic growth phase (OD600=0.4-0.7).The E. coli is cultured at 37° C. while gently stirring for one hour toallow phages to infect the E. coli. The infected E. coli are seeded on225 mm×225 mm plates. Then, phages are collected from the culturesolution of the seeded E. coli to collect a phage library suspension.Similar panning is performed several times to obtain a population ofphages that bind to adenosine, AMP, ADP, or ATP.

Since phages that bind to an antigen (adenosine, AMP, ADP, or ATP)obtained from each of the heavy chain and light chain variable regionphage-display libraries are a population that has antigen-bindingability, an Fab-displaying phage library constructed by combining thetwo is predicted to contain a large number of clones that retain thebinding to small molecules serving as a switch. Thus, it is thought thata library that allows isolation of switch antibodies with higherefficiency can be generated.

Genes are extracted by methods known to those skilled in the art from E.coli infected with each of the phage libraries of the heavy chain andlight chain. Using as template the collection (library) of each of thegenes obtained as described above, gene libraries are amplified withprimers capable of amplifying each of VH and VL. The gene library of theamplified human antibody heavy chain variable region and the genelibrary of the amplified human antibody light chain variable region areintroduced into an appropriate phagemid vector having both a humanIgG-derived CH1 sequence and a human IgG-derived light chain constantregion sequence. The phagemid vectors are introduced into E. coli byelectroporation to construct a design library which enables to obtainantibodies presenting Fab domains consisting of a human antibodyvariable region-constant region and which bind to antigens usingadenosine, AMP, ADP, or ATP as a switch. A design library composed of Hchains and L chains having such diverse binding activity to adenosine,AMP, ADP, or ATP is thought to be useful as a library containing humanantibodies that enable to efficiently obtain AMP/ADP/ATP/adenosineswitch antibodies against arbitrary antigens, where adenosine, AMP, ADP,or ATP is sandwiched between the antibody and antigen as shown in FIG.43. Moreover, since, as described above, SMB0002 binds not only toadenosine and AMP but also to ADP and ATP, it is also predicted to havebinding activity to cAMP and ATP-gamma-S which are structurally similarto AMP, ADP, ATP, and adenosine. Thus, the library is considered to beuseful for obtaining switch antibodies whose binding activity toward anarbitrary target antigen varies depending on the presence of any one ormore small molecules of ATP, ADP, AMP, cAMP, adenosine, and ATP-gamma-S.

Example 6 Acquisition of Antibodies that Bind to the Human IL-6 Receptor(hIL-6R) in the Presence of Small Molecules from a Human AntibodyLibrary Using the Phage Display Technique (6-1) Acquisition ofAntibodies that Bind to hIL-6R in the Presence of Small Molecules from aNaive Human Antibody Library by Bead Panning

A naïve human antibody phage-display library constructed as described inReference Example 1 described below was screened for antibodies showinghuman IL-6 receptor (hIL-6R)-binding activity in the presence of smallmolecules. Specifically, phages presenting antibodies showing bindingactivity toward hIL-6R captured onto beads in the presence of smallmolecules were collected. Phages were collected from the phage elutionsolution eluted from the beads in the absence of the small molecules. Inthis acquisition method, biotin-labeled hIL-6R was used as antigen.

Phages produced by E. coli retaining the constructed phagemids for phagedisplay were purified by a common method. Then, a phage librarysuspension dialyzed against TBS was obtained. Next, BSA was added at afinal concentration of 4% to the phage library suspension. Panning wasperformed using antigens immobilized onto magnetic beads. The magneticbeads used were NeutrAvidin coated beads (Sera-Mag SpeedBeadsNeutrAvidin-coated) or Streptavidin coated beads (Dynabeads M-280Streptavidin).

To efficiently isolate small molecule switch antibodies dependent onsmall molecules that can play a role as a switch in cancer tissues,panning was carried out, which concentrates antibodies that bind toantigens in the presence of a mixed solution of the following varioussmall molecules (adenosine, adenosine 5′-triphosphate (ATP), inosine,kynurenine, prostaglandin E2 (PGE2), succinic acid, and lactic acid)(hereinafter referred to as small molecule cocktail (SC)), but do notbind to antigens in the absence of SC.

Specifically, SC was prepared to contain adenosine triphosphate sodiumsalt (ATP-Na), adenosine, inosine, succinic acid, and lactic acid, eachat a final concentration of 1 mM, prostaglandin E2 (PGE2) at a finalconcentration of 1 μM, and kynurenine at a final concentration of 100μM, and its pH was adjusted to 7.4 with NaOH. The prepared phage librarysuspension was contacted with the SC and 250 pmol of biotin-labeledantigens at room temperature for 60 minutes. Then, BSA-blocked magneticbeads were added to the phage library suspension, and the antigen/phagecomplexes were allowed to bind to the magnetic beads at room temperaturefor 15 minutes. The beads were washed once with SC/TBS (TBS containingSC). Then, beads to which 0.5 mL of 1 mg/mL trypsin solution has beenadded were suspended for 15 minutes at room temperature, and a phagesuspension was collected from the beads separated immediately thereafterusing a magnetic stand. The collected phage suspension was added to 10ml of E. coli strain ER2738 in a logarithmic growth phase(OD600=0.4-0.7). The E. coli was cultured at 37° C. while gentlystirring for one hour to allow phages to infect the E. coli. Theinfected E. coli were seeded on 225 mm×225 mm plates. Then, phages arecollected from the culture solution of the seeded E. coli to collect aphage library suspension.

The first panning was carried out to collect phages that can bind in thepresence of small molecules, while the second and subsequent panningswere carried out to concentrate phages that can bind to antigens only inthe presence of SC. Specifically, 40 pmol of biotin-labeled antigen, SC,and NaOH were added to the prepared phage library suspension to let thephage library contact with the antigen and small molecules at roomtemperature for 60 minutes. BSA-blocked magnetic beads were added, andthe antigen/phage complex was allowed to bind to the magnetic beads atroom temperature for 15 minutes. The beads were washed with 1 mL ofSC/TBST and SC/TBS. Then, beads to which 0.5 mL of TBS has been addedwere suspended at room temperature, and a phage suspension was collectedfrom the beads separated immediately thereafter using a magnetic stand.This process was repeated, and then the two phage suspensions separatelyeluted were combined. 5 μL of 100 mg/mL trypsin was added to thecollected phage suspension to cleave the pIII protein of phages that donot display Fab (helper phage-derived pIII protein), resulting in lossof the ability of phages not displaying Fab to infect E. coli. Thephages collected from the trypsin-treated phage suspension were added to10 mL of E. coli strain ER2738 in a logarithmic growth phase(OD600=0.4-0.7). The E. coli was cultured at 37° C. while gentlystirring for one hour to allow phages to infect the E. coli. Theinfected E. coli was seeded on 225 mm×225 mm plates. Then, phages werecollected from the culture solution of the seeded E. coli to collect aphage library suspension. Three rounds of panning were performed in thepresence of SC to obtain antibodies having antigen-binding activity.

(6-2) Assessment of Binding Activity in the Presence of Small Moleculesby Phage ELISA

From a single colony of E. coli obtained in (6-1), a phage-containingculture supernatant was collected according to a conventional method(Methods Mol. Biol. (2002) 178, 133-145). Purified phages that werepurified by the method described in Reference Example (1-3) weresubjected to ELISA by the procedure described below. A StreptaWell 96microtiter plate (Roche) was coated overnight with 100 μL of TBScontaining biotin-labeled hIL-6R. After removing biotin-labeled hIL-6Rthat did not bind to the plate by washing each of the wells of the platewith TBST, the wells were blocked with 250 μL of 2% skim milk/TBS forone hour or longer. After removing 2% skim milk/TBS, purified phageswere added to each well of the plate, and the plate was left still atroom temperature for one hour to allow antibody-presenting phages tobind to biotin-labeled hIL-6R present in each well in thepresence/absence of SC. Each well was washed with TBST or SC/TBST, andHRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) dilutedwith TBS or SC/TBS was added thereto, and the plate was incubated forone hour. After washing with TBST or SC/TBST, the chromogenic reactionof TMB single solution (ZYMED) added in each well was terminated byadding sulfuric acid. Then, the color development was measured byabsorbance at 450 nm.

Clones 6RNMSC1-2_F02 and 6RNMSC1-3_G02 having binding activity to theantigen hIL-6R in the presence of a small molecule mixture were obtainedby carrying out phage ELISA using 960 isolated clones.

(6-3) Expression and Purification of Antibodies that Bind to hIL-6R

Using specific primers (SEQ ID NOs: 78 and 80), genes were amplifiedfrom clones 6RNMSC1-2_F02 and 6RNMSC1-3_G02 determined to have bindingactivity to biotin-labeled hIL-6R in the presence of SC based on thephage ELISA described in (6-2). The nucleotide sequences of the geneswere analyzed (6RNMSC1-2_F02: the heavy chain sequence is shown in SEQID NO: 32 and the light chain sequence is shown in SEQ ID NO: 33;6RNMSC1-3_G02: the heavy chain sequence is shown in SEQ ID NO: 34 andthe light chain sequence is shown in SEQ ID NO: 35). The genes encodingthe variable regions of 6RNMSC1-2_F02, 6RNMSC1-3_G02, and the negativecontrol anti-human glypican 3 antibody GC413 (heavy chain: SEQ ID NO:36; light chain: SEQ ID NO: 37) were inserted into an animal expressionplasmid carrying human IgG1/Kappa. The expressed antibodies werepurified by the method described in Reference Example 1 described below.

(6-4) Identification of Small Molecules that are Required for hIL-6RBinding of Isolated Antibodies

Three types of obtained antibodies 6RNMSC1-2_F02, 6RNMSC1-3_G02, andGC413 were subjected to ELISA under the nine conditions shown in Table12. Meanwhile, small molecules were suitably prepared at theconcentrations shown in Table 12 using the buffers listed in Table 13.The antigen used was biotin-labeled hIL-6R.

TABLE 12 Condition Small molecule Concentration 1 ATP-Na 1 mM 2Adenosine 1 mM 3 Inosine 1 mM 4 PGE2 1 μM 5 Succinic acid 1 mM 6 Lacticacid 1 mM 7 Kynurenine 100 μM 8 ATP 1 mM, Adenosine 1 mM, Inosine 1 mM,PGE2 1 μM, Succinic acid 1 mM, Lactic acid 1 mM, Kynurenine 100 μM 9 — —

TABLE 13 Wash buffer 10 mM ACES, 150 mM NaCl, 0.05% Tween20, pH 7.4Blocking Buffer 10 mM ACES, 150 mM NaCl, 2% SkimMilk, pH 7.4 SampleBuffer 10 mM ACES, 150 mM NaCl, Small molecule, pH 7.4

First, a StreptaWell 96 microtiter plate (Roche) was coated with 100 μLof PBS containing biotin-labeled hIL-6R at room temperature for one houror longer. After removing biotin-labeled hIL-6R that did not bind to theplate by washing each well of the plate with TBST, the wells wereblocked with 250 μl of Blocking Buffer (2% skim milk/TBS) for one houror longer, and Blocking Buffer was removed from each well. 100 μL ofeach of the purified IgGs prepared at 2.5 μg/mL using Sample Buffercontaining small molecules at the final concentrations shown in Table 12were added to each well, and the plate was let still at room temperaturefor one hour to allow each IgG to bind to biotin-labeled hIL-6R presentin each well. After washing with Wash Buffer containing small moleculesat the final concentrations shown in Table 12, HRP-conjugated anti-humanIgG antibody (BIOSOURCE) diluted with Sample Buffer containing the samesmall molecules was added to each well and the plate was incubated forone hour. After washing with Wash Buffer containing respective smallmolecules, the chromogenic reaction of TMB single solution (ZYMED) addedin each well was terminated by adding sulfuric acid. Then, the colordevelopment was measured by absorbance at 450 nm. The composition of theBuffer used is shown in Table 13.

The measurement results are shown in FIGS. 16 and 17. When 6RNMSC1-2_F02and 6RNMSC1-3_G02 were used, the obtained result was that the absorbanceunder condition 9 (no small molecule) was significantly lower ascompared to the absorbance under condition 8 (cocktail solutioncontaining all of the small molecules). It was confirmed from thisresult that 6RNMSC1-2_F02 and 6RNMSC1-3_G02 had the property that theirantigen binding varies depending on the presence or absence of the smallmolecules. Moreover, the result obtained when 6RNMSC1-2_F02 was used wasthat the absorbance under condition 7 (100 uM kynurenine) was comparableto that under condition 8, but the absorbance was significantly lowunder other conditions; thus, it was demonstrated that 6RNMSC1-2_F02 isan antibody that binds to the antigen hIL-6R in the presence ofkynurenine (FIG. 16). Further, the result obtained when 6RNMSC1-3_G02was used was that the absorbance under condition 1 (1 mM ATP-Na) wascomparable to that under condition 8, but the absorbance wassignificantly low under other conditions; thus, it was demonstrated that6RNMSC1-3_G02 is an antibody that binds to the antigen hIL-6R in thepresence of ATP (FIG. 17). It was shown that several antibodies whoseantigen-binding ability varies under the presence of different smallmolecules could be isolated at one time by using a method as describedabove.

Example 7 Characterization of Antibody 6RNMSC1-2_F02 (7-1) Assessment byELISA of hIL-6R-Binding Activity in the Presence of Amino Acids andAmino Acid Metabolites Other than Kynurenine

Antibody 6RNMSC1-2_F02 obtained in Example 6, which binds to hIL-6R inthe presence of small molecules, is an antibody that binds to hIL-6R inthe presence of kynurenine. A series of amino acid metabolites such astryptophan metabolites were assessed for whether they are appropriate asa non-limiting embodiment of cancer tissue-specific compounds, inparticular, cancer cell-specific metabolites, for use in the presentinvention.

Antibody 6RNMSC1-2_F02 having antigen-binding activity in the presenceof kynurenine described in Example 6 and negative control GC413 weresubjected to ELISA under the seven conditions described in Table 14.Meanwhile, each of the amino acids and metabolites thereof wereappropriately prepared at the concentrations shown in Table 14 using thebuffers shown in Table 13. The antigen used was biotin-labeled hIL-6R.

TABLE 14 Condition Small Molecule Concentration 1 Kynurenine 1 mM 2Tryptophan 1 mM 3 Phenylalanine 1 mM 4 Anthranilic acid 1 mM 53-Hydroxykynurenine 1 mM 6 Kynurenic acid 1 mM 7 — —

First, a StreptaWell 96 microtiter plate (Roche) was coated with 100 μLof PBS containing biotin-labeled antigens at room temperature for onehour or longer. After removing antigens that did not bind to the plateby washing each well of the plate with TBST, the wells were blocked with250 μl of Blocking Buffer (2% skim milk/TBS) for one hour or longer, andBlocking Buffer was removed from each well. 100 μL of each of thepurified IgGs prepared at 2.5 μg/mL using Sample Buffer containing smallmolecules at the final concentrations shown in Table 14 were added toeach well, and the plate was let still at room temperature for one hourto allow each IgG to bind to the antigen present in each well. Afterwashing with Wash Buffer containing amino acids and amino acidmetabolites at the final concentrations shown in Table 14,HRP-conjugated anti-human IgG antibody (BIOSOURCE) diluted with SampleBuffer containing amino acids and amino acid metabolites was added toeach well, and the plate was incubated for one hour. After washing withWash Buffer containing the respective amino acids and amino acidmetabolites, the chromogenic reaction of TMB single solution (ZYMED)added in each well was terminated by adding sulfuric acid. Then, thecolor development was measured by absorbance at 450 nm. The compositionof the Buffer used is shown in Table 13.

The measurement results are shown in FIG. 18. When 6RNMSC1-2_F02 wasused, the absorbance under condition 7 (no small molecule) wassignificantly lower as compared to the absorbance under condition 1(kynurenine solution). Similarly, the absorbance under condition 5(3-hydroxy kynurenine solution) showed a high absorbance similarly tothat under condition 1; thus, it was shown that 6RNMSC1-2_F02 is anantibody that binds to the antigen hIL-6R not only in the presence ofkynurenine but also in the presence of a kynurenine metabolite.Moreover, since the absorbance was significantly low under otherconditions, 6RNMSC1-2_F02 was shown to be an antibody that does not bindto the antigen hIL-6R even if tryptophan, a precursor of kynurenine, ispresent. Since the expression of IDO, which is an enzyme that produceskynurenine by metabolizing tryptophan, is elevated in cancermicroenvironments, antibodies that bind to antigens in the presence ofkynurenine or metabolites thereof but not in the presence of tryptophanare considered to be important as antibodies that bind to antigens onlyin cancer microenvironments. Furthermore, from the above, it was thoughtthat the same method can be used to obtain antibodies that bind to anantigen of interest not only in the presence of a single type of aminoacid metabolite, but also in the presence of multiple types ofstructurally different amino acid metabolites.

Thus, an antibody which uses as a switch a molecule that is producedlocally in cancer via metabolism by cancer-specific enzymes (in thisExample, IDO and its downstream metabolic enzymes) is thought to be ableto selectively bind to target antigens only locally in cancer. In thisExample, metabolic products generated from the endogenous moleculetryptophan by cancer-specific metabolic enzymes are used as a switch;however, it is considered that it is possible to use, as a switch, anartificial molecule (unnatural molecule) that is administeredexogenously such as by oral or intravenous administration as well as ametabolic product generated from an endogenous molecule bycancer-specific metabolic enzymes. For example, as described in Example1, capecitabine (xeloda), when administered, is metabolized into 5-FU bycancer-specific metabolic enzymes or such, resulting in an increase oflocal 5-FU concentration in cancer (Desmoulin F. et al., Drug MetabDispos. 2002). Thus, it is considered that an antibody that uses 5-FU asa switch can selectively bind to the target antigen only locally incancer. Alternatively, it is considered possible to use, as a switch,molecules produced due to cancer-specific hypoxic environments or acidicenvironments, instead of metabolic enzymes. For example, since, underhypoxic conditions, TH-302 (Duan J X, et al., J Med Chem. 2008) ismetabolized into Br-IPM, an antibody that uses Br-IPM as a switch couldselectively bind to target antigens only locally in cancer.

(7-2) Assessment by Surface Plasmon Resonance for the Effect ofKynurenine on Human IL6 Receptor Binding

Biacore T200 (GE Healthcare) was used to analyze the interaction ofantigen-antibody reaction between 6RNMSC1-2_F02 and human IL-6 receptor(hIL-6R). An appropriate amount of protein A (Invitrogen) wasimmobilized onto Sensor chip CM5 (GE Healthcare) by the amine couplingmethod. The antibody of interest was captured onto the sensor chip, andthe antigen hIL-6R was allowed to interact. The running buffer used was20 mmol/L ACES, 150 mmol/L NaCl, 0.05% (w/v) Tween 20, pH 7.4. Theinteraction with the antigen hIL-6R was measured at 25° C. hIL-6R wasdiluted with the running buffer, the running buffer containing 100μmol/L kynurenine, or, as a control, the running buffer containing 10mmol/L ATP.

Diluted hIL-6R solutions and the running buffer which is the blank wereinjected at a flow rate of 10 μL/min for one minute and hIL-6R wasallowed to interact with 6RNMSC1-2_F02 captured on the sensor chip.Then, the running buffer was run at a flow rate of 10 μL/min for oneminute to observe the dissociation of hIL-6R from the antibody. Next, 10mmol/L glycine-HCl, pH 1.5, was injected at a flow rate of 30 μL/min for30 seconds to regenerate the sensor chip. The dissociation constantK_(D) (M) of 6RNMSC1-2_F02 for hIL-6R was calculated from the kineticparameters, the association rate constant ka (l/Ms) and the dissociationrate constant kd (l/s), calculated based on the sensorgrams obtained bythe measurements. Each parameter was calculated using Biacore T200Evaluation Software (GE Healthcare).

Sensorgrams obtained by this measurement of the interaction between6RNMSC1-2_F02 and 1 μmol/L hIL-6R in the presence of 100 μmol/Lkynurenine, 10 mmol/L ATP, or in the absence of these are shown in FIG.19. As shown in FIG. 19, in the presence of 100 μmol/L kynurenine,6RNMSC1-2_F02 bound to hIL-6R; however, hIL-6R binding was not detectedin the absence of kynurenine. Thus, 6RNMSC1-2_F02 was confirmed to havethe property of binding to hIL-6R using kynurenine as a switch. Further,the dissociation constant KD of 6RNMSC1-2_F02 in the presence of 100μmol/L kynurenine was 1.5 μmol/L.

(7-3) Effect of Kynurenine as a Switch on Dissociation of the Antibodyfrom hIL-6R

Whether 6RNMSC1-2_F02 that bound to hIL-6R in the presence of kynureninedissociates in the presence of kynurenine in a kynurenineconcentration-dependent manner was assessed using Biacore T200 (GEHealthcare). The running buffers used were 20 mmol/L ACES, 150 mmol/LNaCl, 0.05% (w/v) Tween 20, pH 7.4, and 20 mmol/L ACES, 150 mmol/L NaCl,0.05% (w/v) Tween 20, pH 7.4, 100 μmol/L kynurenine. The measurementswere performed at 25° C. As an analyte, 5 μg/mL 6RNMSC1-2_F02 dilutedwith 20 mmol/LACES, 150 mmol/L NaCl, 0.05% (w/v) Tween 20, pH 7.4,containing 100 μmol/L kynurenine was allowed to interact for 180 secondswith hIL-6R immobilized onto Sensor chip CM5 by amine coupling. Then,the mode of the dissociation of hIL-6R was observed under each runningbuffer condition. In order to compare the extent of dissociation undereach running buffer condition, the amount of 6RNMSC1-2_F02 bound tohIL-6R in the presence of 100 μmol/L kynurenine was taken as 100 fornormalization, and the normalized values were compared. The sensorgramshowing the interaction between 6RNMSC1-2_F02 and hIL-6R afternormalization is shown in FIG. 20. The result of FIG. 20 revealed that,after binding to hIL-6R in the presence of kynurenine, 6RNMSC1-2_F02 hadthe property of rapidly dissociating hIL-6R when kynurenine is no morepresent. Specifically, the regulation by kynurenine on binding of theantibody to hIL-6R was confirmed to be reversible.

(7-4) Assessment of the Effect by Kynurenine Concentration on hIL-6RBinding

Next, the effect of kynurenine concentration on antigen-antibodyreaction between 6RNMSC1-2_F02 and hIL-6R was assessed using BiacoreT200 (GE Healthcare). The running buffer used was 20 mmol/L ACES, 150mmol/L NaCl, 0.05% (w/v) Tween 20, pH 7.4. Antigen-antibody reactionbetween 6RNMSC1-2_F02 and human IL-6R was assayed at 25° C. hIL-6R wasimmobilized onto Sensor chip CM5 by amine coupling. As an analyte, 1μg/mL 6RNMSC1-2_F02 diluted with 20 mmol/L ACES, 150 mmol/L NaCl, 0.05%(w/v) Tween 20, pH 7.4, containing kynurenine adjusted to variousconcentrations were allowed to interact for 180 seconds and changes inthe amount of binding were observed. The result is shown in FIG. 21.From this result, it was revealed that the higher the concentration ofkynurenine acting as a switch, the greater the amount of 6RNMSC1-2_F02binding to hIL-6R.

Since, in this assay system, hIL-6R is immobilized on a sensor chip,6RNMSC1-2_F02 is thought to bind in a divalent manner. The amount of6RNMSC1-2_F02 that bound to hIL-6R was also observed to increase whenthe kynurenine concentration increases in this assay system where6RNMSC1-2_F02 recognizes hIL-6R in a divalent manner. This result showedthat, in divalent binding as well, 6RNMSC1-2_F02 has the property ofbinding to IL-6R using kynurenine as a switch.

The results described above revealed that 6RNMSC1-2_F02 is an antibodywhich, using kynurenine as a switch, binds to hIL-6R in the presence ofkynurenine and dissociates from hIL-6R in the absence of kynurenine.Furthermore, 6RNMSC1-2_F02 was confirmed not to show hIL-6R-bindingactivity in the absence of kynurenine, enabling a complete ON/OFFregulation, and to fulfill a switch function.

(7-5) Assessment of the Binding Ability of 6RNMSC1-2_F02 for KynurenineDerivatives

Kynurenine-dependent antibody 6RNMSC1-2_F02 obtained from the librarywas assessed for its binding to kynurenine and derivatives thereof usingBiacore T200 (GE Healthcare). The following seven molecules wereassessed as derivatives: the kynurenine isomer D-kynurenine and thederivatives 3-hydroxy-DL-kynurenine, 4-(4-methylphenyl)-4-oxobutyricacid, R00447436-000-001, RO0635389-000-001, R00438566-001-001, andRO0635390-000-001. The chemical structure of each compound is shownbelow.

An appropriate amount of protein A (Invitrogen) was immobilized ontoSensor chip CM4 (GE Healthcare) by the amine coupling method. Theantibody of interest was captured thereon, and the antigen kynurenineand derivatives thereof were allowed to interact. The running bufferused was 50 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.02% (w/v) Tween 20, 5%DMSO, pH 7.6. All measurements were carried out at 15° C. The antigenswere diluted with the running buffer.

Regarding 6RNMSC1-2_F02, the diluted antigen solutions and the runningbuffer as the blank were each added at a flow rate of 30 μL/min for 60seconds to allow each antigen to interact with the antibody captured onthe sensor chip. Then, the running buffer was run at a flow rate of 30μL/min for 60 seconds to observe the dissociation of antigens from theantibody. Kinetic parameters, the association rate constant ka (l/Ms)and the dissociation rate constant kd (l/s), were calculated based onthe sensorgrams obtained by the measurements. The dissociation constantKD (M) was calculated from these constants. Each parameter wascalculated using Biacore T200 Evaluation Software (GE Healthcare).

Based on the assessment result, the kinetic parameters of theinteraction between kynurenine and 6RNMSC1-2_F02 were determined as:ka=709 (l/s), kd=0.17 (l/s), and KD=0.239 (mmol/L). Sensorgrams obtainedas a result of the assessment are shown in FIG. 22. Sensorgramsrepresenting the results of the binding with the derivatives aredepicted in: FIG. 23 for 3-hydroxy-DL-kynurenine, FIG. 24 forRO0635389-000-001, and FIG. 25 for RO0635390-000-001. Scrubber2(Biologic software) and Microsoft Office Excel 2007 were used to showthe sensorgrams. These results revealed that 6RNMSC1-2_F02 can not onlybind to kynurenine, but also to some kynurenine derivatives such as3-hydroxy-DL-kynurenine, RO0635389-000-001, and RO0635390-000-001.

(7-6) Assessment of Responsiveness of 6RNMSC1-2_F02 to Unnatural SmallMolecules

Octet (PRIMETECH) was used to assess the effect of various smallmolecules on the antigen-antibody reaction between hIL-6R and thekynurenine-switch antibody 6RNMSC1-2_F02 obtained from a library. Atotal of eight molecules shown in Example (7-5), which are kynurenineand derivatives thereof, were evaluated as small molecules. The assaybuffer used was TBS, pH 7.4, and measurements were carried out at 30° C.Biotinylated hIL-6R was immobilized onto a streptavidin sensor chip, theantibody was allowed to interact, and changes in the amount of itsbinding were observed. The antibody was diluted using the assay bufferor the assay buffer to which either one of the small molecules has beenadded. The final concentration of each small molecule was adjusted to100 uM, while the final antibody concentration was adjusted to 10 μg/mL.

The hIL-6R binding activity of 6RNMSC1-2_F02 obtained by thismeasurement in the presence of each small molecule at 100 uM or in theirabsence is shown in: FIG. 26 for kynurenine, FIG. 27 for3-hydroxy-DL-kynurenine, FIG. 28 for RO0635389-000-001, and FIG. 29 forRO0635390-000-001. As shown in FIG. 26, in the presence of 100 uMkynurenine, 6RNMSC1-2_F02 bound to hIL-6R, while in the absence ofkynurenine, hIL-6R binding was not observed. Thus, Octet also confirmedthat 6RNMSC1-2_F02 had the property of binding to hIL-6R usingkynurenine as a switch. Furthermore, as shown in FIGS. 27 to 29, bindingto hIL-6R was observed in the presence of the small molecules other thankynurenine such as 3-hydroxy-DL-kynurenine, RO0635389-000-001, andRO0635390-000-001 which are kynurenine derivatives capable of binding to6RNMSC1-2_F02, while in the absence of these small molecules, binding tohIL-6R was not observed. The above demonstrated that anti-hIL-6Rantibodies for which kynurenine and derivatives thereof (unnaturalcompounds) function as a switch can be isolated from a library.

Example 7-3 showed the utilization of endogenous molecules as a switch.With this Example, it has been shown that artificial molecules(unnatural molecules) can also be used as a switch. Specifically, it hasbeen demonstrated that antibodies can be generated, which use as aswitch unnatural metabolites produced by cancer-specific metabolicenzymes from artificial molecules (unnatural molecules) that can beadministered exogenously by oral or intravenous routes or such.

Example 8 Design of Library for Obtaining Kynurenine-Switch Antibodiesby Comprehensive Alteration Using a Kynurenine-Switch Anti-hIL-6RAntibody

The concentration of kynurenine is known to be high in cancer tissues.Several antibodies showing antigen-binding ability only in the presenceof ATP were obtained from a rationally designed library constructed inReference Example 2 using as a template an ATP-binding antibody. Thissuggested that antibodies showing antigen-binding ability only in thepresence of kynurenine could also be obtained by constructing a libraryusing as a template an antibody that shows binding ability tokynurenine.

(8-1) X-Ray Crystallographic Structure Analysis of the hIL-6R-BindingAntibody 6RNMSC1-2_F02 which Uses Kynurenine as a Switch

The three-dimensional structure of the complex of kynurenine andantibody 6RNMSC1-2_F02 that binds to hIL-6R using kynurenine as aswitch, which was obtained from a library in Example 7, was revealed byx-ray crystallographic structure analysis.

(8-1-1) Preparation of the Full-Length 6RNMSC1-2_F02 Antibody forCrystallization

The full-length 6RNMSC1-2_F02 antibody for crystallization was preparedand purified by a method known to those skilled in the art.

(8-1-2) Preparation of 6RNMSC1-2_F02 Fab Fragment from the Full-LengthAntibody

After the obtained antibody 6RNMSC1-2_F02 was concentrated with 10000molecular weight cutoff (MWCO) ultrafiltration membrane, a sample wasprepared by diluting the antibody to 2 mg/ml with 100 mM Tris buffer, pH8.0, and Endoproteinase Lys-C Sequencing Grade (Roche Applied Science)was added thereto at a mass ratio of 1/400 to the full-length antibody.The mixture was incubated at 35° C. for 45 minutes. Then, the reactionwas terminated by adding 20 ml of 25 mM sodium acetate buffer, pH 5.0into which a tablet of protease inhibitor cocktail mini, EDTA-free(Roche Applied Science) has been dissolved. Next, this sample was addedto a 1-ml size cation-exchange column HiTrap SP HP (GE Healthcare) towhose downstream 1-ml size Protein A-carrying column HiTrap MabSelectSure (GE Healthcare) was tandemly connected and which was equilibratedwith 25 mM sodium acetate buffer, pH 5.0. Elution was performed bylinearly increasing the concentration of NaCl in the buffer, and apurified fraction of Fab fragments of the 6RNMSC1-2_F02 antibody wasobtained. This was added to the gel filtration column Superdex 200 16/60prep grade (GE Healthcare) equilibrated with 25 mM HEPES buffer, pH 7.5,100 mM NaCl. The Fab fragments of 6RNMSC1-2_F02 were eluted with thesame buffer from this column for crystallization. All column operationswere carried out at low temperature.

(8-1-3) Preparation of Crystals of the Complex of Kynurenine and6RNMSC1-2_F02 Fab Fragment

A sample of 6RNMSC1-2_F02 Fab fragment for crystallization purified bythe above-described method was concentrated with a 5000 MWCOultrafiltration membrane to A280=24.1. Then, 50 mM kynurenine dissolvedin 100% DMSO was added at a final concentration of 2 mM, andcrystallization was carried out using the sitting drop vapor diffusionmethod. Crystallization was carried out using Hydra II Plus One(MATRIX). Using a reservoir solution of 0.2 M lithium sulfatemonohydrate, 30.0% w/v PEG 3350, and 0.1 M Tris, pH 8.5, crystallizationdrops were prepared by mixing at reservoir solution:crystallizationsample=0.2 μl:0.2 The drops were allowed to stand at 20° C. and thincolumnar crystals were successfully obtained. Then, one of the obtainedcrystals was immersed in a 0.18 M lithium sulfate monohydrate, 27.3%PEG3350, 0.09 M HEPES pH7.5, 18.2% ethylene glycol, 4.5 mM kynurenine,9% DMSO solution for 30 minutes at room temperature, and a crystal ofthe complex of 6RNMSC1-2_F02 Fab fragment and kynurenine was obtained.

(8-1-4) Measurement of X-Ray Diffraction Data from the Crystal of theComplex of 6RNMSC1-2_F02 Fab Fragment and Kynurenine

A single crystal of the complex of 6RNMSC1-2_F02 Fab fragment andkynurenine prepared by the method described above was scooped togetherwith the solution using a pin equipped with a minute nylon loop, andfrozen in liquid nitrogen. X-ray diffraction data were measured usingBL-5A at the synchrotron radiation facility Photon Factory of the HighEnergy Accelerator Research Organization. The frozen state wasmaintained through the measurement by placing in a stream of nitrogengas at −178° C. A total of 360 X-ray diffraction images were collectedusing CCD detector Quantum 315r (ADSC) attached to the beamline byrotating the crystal by 0.5°. Lattice constant determination,diffraction spot indexing, and diffraction data processing from theobtained diffraction images were performed using the programs Xia2 (J.Appl. Cryst. (2010) 43, 186-190), XDS Package (Acta Cryst. (2010) D66,125-132), and Scala (Acta Cryst. (2006) D62, 72-82). Ultimately, thissuccessfully yielded diffraction intensity data of up to 2.33 angstromresolution. This crystal belonged to space group P1 with latticeconstants a=55.830 angstrom, b=56.040 angstrom, c=80.340 angstrom,α=87.81°, β=82.88°, γ=65.54°.

(8-1-5) X-Ray Crystallographic Analysis of the Complex of Kynurenine and6RNMSC1-2_F02 Fab Fragment

To determine the structure of the complex of kynurenine and6RNMSC1-2_F02 Fab fragment, the molecular replacement method was carriedout using the program Phaser (J. Appl. Cryst. (2007) 40, 658-674). Thenumber of complexes in the asymmetrical unit was estimated to be twofrom the size of the obtained crystal lattice and the molecular weightof the 6RNMSC1-2_F02 Fab fragment. A homology model of the antibody wasconstructed using Discovery Studio3.5 (Accelrys). This Fab model wasdivided into the variable region and constant region, and using thecoordinate of each structure as search model, their orientation andposition in the crystal lattices were determined based on the rotationfunction and translation function. Further, the crystallographicreliability factor R for the diffraction intensity data at 25 to 3.0angstroms was 40.57% and Free R was 38.91%, when rigid body refinementwas carried out on the obtained initial structural model in which thevariable region and constant region portions were independently moved.Then, structural model refinement was carried out by repeating thefollowing processes: structural refinement using the program REFMAC5(Acta Cryst. (2011) D67, 355-367), and revision of the structural modelperformed using the program Coot (Acta Cryst. (2010) D66, 486-501) byreferring to the electron density maps having as coefficients 2Fo-Fc andFo-Fc, which were calculated based on the experimentally determinedstructural factor Fo, the structural factor Fc calculated from themodel, and the phase calculated from the model. Ultimately, with 34135diffraction intensity data at 25 to 2.33 angstrom resolution, thecrystallographic reliability factor R and Free R for the structuralmodel containing 7019 non-hydrogen atoms were 20.4% and 26.56%,respectively.

(8-1-6) Identification of the Interaction Sites of 6RNMSC1-2_F02 andKynurenine

Ultimately, the crystallographic structure of the complex betweenkynurenine and the Fab fragment of antibody 6RNMSC1-2-F02 that binds tohIL-6R using kynurenine as a switch, which was obtained from a libraryin Example 7, was determined at a resolution of 2.33 angstrom.

There were two Fab fragment molecules of the antibody in theasymmetrical unit of the crystal, and kynurenine was bound to only oneof them. It was revealed that kynurenine was recognized mainly by theH-chain CDR3 of the antibody, and bound to the antibody at a positionslightly deviated from the part usually used in antigen binding.

As shown in FIG. 30, a kynurenine molecule is recognized by therespective side chains of H-chain P97, R100c, and D101, and L-chain H49and F55, as well as by the respective main chains of H-chain R94, D95,R100c, G100d, and A100e of the antibody. Furthermore, the amino groupsin the amino acid backbone of kynurenine are positively charged atneutral pH, and several hydrogen bonds and electrostatic interactionsare observed with the main chain carbonyl oxygen of each of the H-chainD95, R94, and A100e, and the side chain carboxy group of D101. Moreover,the negatively charged carboxy group of kynurenine forms severalhydrogen bonds with the amide NH group of the main chain of each of theH-chain G100d and R100c. The network of these strong hydrogen bonds andelectrostatic interactions is expected to play an important role inrecognition of kynurenine by the antibody. Furthermore, the side chainof H-chain R100c forms a cation-π interaction with the benzene ringmoiety of kynurenine. In addition, the L-chain H49 and F55 and H-chainD101 surround the benzene ring moiety of kynurenine, and formation ofvan der Waals interactions with these residues contribute to bindingrecognition for kynurenine. Further, the side chain of L-chain H49 wasconfirmed to form a hydrogen bond, though weak, with the NH2 group inthe aromatic ring of kynurenine.

The result described above revealed the mode of kynurenine recognitionby the antibody and identified the amino acid residues of the antibodyvariable region that are greatly involved in kynurenine binding. Aminoacid residues whose side chains are significantly involved in kynureninebinding include: P97, R100c, and D101 (Kabat numbering) in the H-chain,and H49 and F55 (Kabat numbering) in the L-chain. Moreover, residueswhose main chain moieties are significantly involved in kynureninebinding include R94, D95, R100c, G100d, and A100e in the H-chain.Further, based on their structural features, residues such as P97,P100b, and G100d are thought to possibly be significantly contributingindirectly to kynurenine binding by maintaining the structure of theH-chain CDR3 in a conformation necessary for binding to kynurenine.

(8-2) Assessment of 6RNMSC1-2_F02 Variants for Binding Ability toKynurenine (8-2-1) Assessment of Heavy Chain Variants of 6RNMSC1-2_F02for Binding Ability to Kynurenine

As described above, by crystallographic structure analysis, heavy-chainP97, P100b, R100c, G100d, D101, and such were found as residues that maybe playing an important role in kynurenine binding. Thus, regarding someof the residues, variants were actually produced to assess their bindingability for kynurenine. The variants constructed are: P97A, P97G,P100bG, R100cA, G100dA, G100dV, and D101A (Table 15).

TABLE 15 Heavy chain Light chain Clone name SEQ ID NO SEQ ID NO6RNMSC1-2_F02 P97A SEQ ID NO: 38 SEQ ID NO: 33 6RNMSC1-2_F02 P97G SEQ IDNO: 39 SEQ ID NO: 33 6RNMSC1-2_F02 P100bG SEQ ID NO: 40 SEQ ID NO: 336RNMSC1-2_F02 R100cA SEQ ID NO: 41 SEQ ID NO: 33 6RNMSC1-2_F02 G100dASEQ ID NO: 42 SEQ ID NO: 33 6RNMSC1-2_F02 G100dV SEQ ID NO: 43 SEQ IDNO: 33 6RNMSC1-2_F02 D101A SEQ ID NO: 44 SEQ ID NO: 33

Various 6RNMSC1-2_F02 variants expressed and purified by the methoddescribed in Reference Example 1 below were assessed for theirkynurenine binding by a similar method as that with Biacore described inExample (7-5). As a result, 6RNMSC1-2_F02 was confirmed once again tobind to kynurenine; however, all the other variants had a reduced orconsiderably reduced ability to bind to kynurenine. Thus, the residuesdeduced to be important for kynurenine binding by the crystallographicstructure were demonstrated to indeed participate in binding, and thisresult supported the results from crystallographic structure analysis.

(8-2-2) Assessment of the H49Y Variant of 6RNMSC1-2_F02 for BindingAbility to Kynurenine

From the crystallographic structure analysis, it was thought that thelight chain amino acid residue that is forming an important interactionwith kynurenine is only H49 (Kabat numbering). Moreover, the result ofsequence analysis suggested that the framework of 6RNMSC1-2_F02 wasderived from VLkappa-2 germline. In VLkappa-2 germline, the amino acidat position 49 by Kabat numbering is Tyr in almost all cases, and ishighly conserved. Thus, a variant was generated by substituting His49with Tyr to assess the degree of contribution of His to the interactionwith kynurenine.

The His49Tyr variant (heavy chain SEQ ID NO: 32; light chain SEQ ID NO:45) of 6RNMSC1-2_F02 expressed and purified by the method of ReferenceExample 1 described below was assessed for kynurenine binding by amethod similar to that with Biacore described in Example (7-5). Theresult revealed that H49Y retained kynurenine binding. Kineticparameters were also determined: ka=2543 (l/s), kd=0.24 (l/s), andKD=0.095 (mmol/L). Sensorgrams showing the assessment results are shownin FIG. 31.

(8-2-3) Modification of 6RNMSC1-2_F02 to a Germline Framework Sequence

The result of sequence analysis suggested that the VH framework of6RNMSC1-2_F02 is derived from a VH1-69 germline while the VL frameworkof 6RNMSC1-2_F02 is derived from a Vκ2-28 germline. Thus, with thepurpose of increasing antibody stability, to restore the frameworksequence of 6RNMSC1-2_F02 to the germline framework sequence, thefollowing modifications were introduced into the framework sequence of6RNMSC1-2_F02: VH_Met108Leu, VL_Thr07Ser, VL_Ser11Leu, VL_Leu15Pro,VL_Gln17Glu, VL_Leu36Tyr, VL_Gln37Leu, VL_Arg39Lys, VL_Pro43Ser,VL_Arg45Gln, VL_His49Tyr, VL_Ala67Ser, and VL_Asn70Asp (numbers indicateKabat numbering). The variant was named F02h011/F02l003 (heavy chainvariable region sequence: SEQ ID NO: 95; light chain variable regionsequence: SEQ ID NO: 96). The Tm of F02h011/F02l003 expressed andpurified by the method described in Reference Example 1-1 was measuredby DSC. Measurement by DSC was carried out by a method known to thoseskilled in the art. The Tm of the variant of 6RNMSC1-2_F02 to whichthese modifications were added increased from 82.9° C. to 85.2° C.,demonstrating stabilization of the structure. Furthermore, kynureninebinding of F02h011/F02l003 was measured by a Biacore-based methoddescribed in Example (7-5), except that the time of adding the dilutedantigen solutions and the running buffer as a blank was modified to 30seconds, and F02h011/F02l003 was confirmed to retain kynurenine binding.Kinetic parameters of kynurenine binding were determined: ka=3664 (l/s),kd=0.40 (l/s), and KD=0.11 (mmol/L). Sensorgrams showing the assessmentresults are shown in FIG. 32. It is sometimes preferable to use highlystable frameworks for antibody libraries; thus, F02h011/F02l003 was usedas the framework sequence in Examples (8-2-3) and (9-2) described below.The framework sequences are shown in Table 16.

TABLE 16 SEQ ID Framework NO Sequence Heavy chain 87 QVQLVQSGAEVKKPGframework 1 SSVKVSCKASGGTFS Heavy chain 88 WVRQAPGQGLEWMG framework 2Heavy chain 89 RVTITADESTSTAYME framework 3 LSSLRSEDTAVYYCAR Heavy chain90 WGQGTLVTVSS framework 4 Light chain 91 DIVMTQSPLSLP framework 1VTPGEPASISC Light chain 92 WYLQKPGQSPQLLIY framework 2 Light chain 93GVPDRFSGSGSGTDFT framework 3 LKISRVEAEDVGVYYC Light chain 94WYLQKPGQSPQLLIY framework 4

(8-2-4) Search for Modifications that Increase the Kynurenine Binding ofF02h011/F02l003

Residues putatively involved in the kynurenine binding of 6RNMSC1-2_F02were predicted based on the results described in Examples (8-1) and(8-2-1). In addition to these residues, a search was carried out forresidues for which an increase in kynurenine binding can be expectedthrough modification.

Specifically, based on the crystallographic structure of the complex ofthe antibody Fab fragment and kynurenine, amino acid residues presentwithin 4.2 angstrom from kynurenine were selected, as shown in FIG. 34.As a result, heavy-chain Tyr32 and Arg100a (Kabat numbering) andlight-chain Leu46 (Kabat numbering) were newly found. Furthermore, sincethe heavy-chain CDR3 is a CDR loop which most highly contributes tokynurenine binding and modification to residues in this loop mayindirectly influence kynurenine binding, amino acid residues weremodified at as many residue positions as possible. However, thefollowing residues were ruled out as a candidate for the modifiedresidue sites: heavy-chain Asp95 which forms a strong hydrogen bond withlight-chain Arg96 and determines the loop structure; heavy-chain Arg100awhich forms a hydrogen bond with light-chain Glu50 and determines theloop structure; and heavy-chain Pro100b whose side chain is oriented inthe opposite direction from kynurenine and for which formation ofinteraction with kynurenine cannot be expected even if a modification isintroduced. Meanwhile, light-chain Ser56, though distant from kynurenineas shown in FIG. 35, was chosen as a site of residue modification, sinceits side chain is oriented in the direction of kynurenine andmodification with an amino acid residue longer than Ser may possiblyresult in formation of an interaction with kynurenine. Furthermore, theresult described in Example (9-2) below showed that kynurenine-bindingactivity was increased by substituting heavy-chain Gly50 and light-chainAsp28 with Ala. Since these residue sites are not positions that candirectly interact with kynurenine (FIG. 36), they are speculated toindirectly contribute by stabilizing the conformation of the overall Fabupon kynurenine binding. Moreover, regarding these residue sites,modification into residues other than Ala can possibly result in furtherincrease of the kynurenine binding; thus, these residue positions werealso chosen as sites for comprehensive alteration. Whether modificationsexpected to result in an effect of increasing the kynurenine-bindingability can be identified was examined by exhaustive assessment ofvariants in which these residues have been modified to various aminoacids.

Such modification sites in F02h011/F02l003 constructed in Example(8-2-3) (positions according to Kabat numbering which are indicated by“Kabat” in the table), the amino acids before modification at thesesites (amino acids indicated by “native sequence” in the table), and theamino acids after modification (amino acids indicated by “altered aminoacids” in the table) are shown in Table 17.

TABLE 17 HCDR1 HCDR2 HCDR3 LCDR1 LFR2 LCDR2 Kabat 32 50 96 97 98 99 100100c 100d 100e 101 102 28 46 49 55 56 Native sequence Y G A P V V A R GA D I D L Y F S Altered A A A A A A A A A A A A A A amino D D D D D D DD D D D D acid E E E E E E E E E E E E E E F F F F F F F F F F F F F F GG G G G G G G G G H H H H H H H H H H H H H I I I I I I I I I I I I I KK K K K K K K K K K K L L L L L L L L L L L L L L L M M M M M M M M M MM M M N N N N N N N N N N N N N N P P P P P P P P P P P Q Q Q Q Q Q Q QQ Q Q Q Q Q R R R R R R R R R R R R S S S S S S S S S S S S S S T T T TT T T T T T T T T V V V V V V V V V V V V V W W W W W W W W W W W W W YY Y Y Y Y Y Y Y Y Y Y Y

The binding of each variant expressed and purified by the methoddescribed in Reference Example 1 described below to kynurenine wasmeasured by the Biacore-based method described in Example (7-5), exceptthat the time of adding the diluted antigen solutions and the runningbuffer as a blank was modified to 30 seconds. Meanwhile, the type ofsensor chip immobilized with Protein A was changed to Sensor chip SeriesS CM3 (GE Healthcare) to measure some of the variants. When kynureninedissociation was too fast to determine the dissociation rate constant, adissociation constant KD (M) was calculated by analysis of equilibriumvalues based on the degree of binding response during the interactionwith each antigen (association phase). In this case also, Biacore T200Evaluation Software (GE Healthcare) was used to calculate theparameters. Based on the assay result, the affinity of each variant forkynurenine was calculated as KD values. The result of comparing the KDvalues of each of the variants and F02h011/F02l003 for kynurenine isshown in Table 18.

TABLE 18 HCDR1 HCDR2 HCDR3 LCDR1 LFR2 LCDR2 Kabat 32 50 96 97 98 99 100100c 100d 100e 101 102 28 46 49 55 56 Native sequence Y G A P V V A R GA D I D L Y F S Altered A 0.0 3.1 0.5 0.1 0.5 0.1 0.1 0.0 0.4 2.5 0.00.1 0.9 amino D 0.0 0.0 0.0 0.0 0.6 0.0 0.0 0.0 0.7 0.0 0.5 acid E 0.00.0 0.4 0.1 0.8 0.0 0.0 0.0 0.5 1.7 0.0 0.0 0.5 F 0.3 1.8 0.8 0.0 0.10.9 0.0 0.0 0.6 2.0 0.0 0.7 1.3 G 0.0 0.0 0.0 1.3 0.0 0.1 1.5 0.0 0.9 H0.4 0.6 1.6 0.0 0.6 2.5 0.1 0.7 2.6 0.1 1.2 1.1 I 0.0 2.7 0.4 0.0 0.21.0 0.0 0.0 0.0 2.1 0.0 1.3 K 0.0 1.9 1.3 0.1 0.6 0.7 0.0 0.2 1.8 0.01.0 L 0.0 1.0 0.1 0.0 0.2 1.2 0.0 0.0 0.0 0.3 0.0 0.0 0.3 0.8 M 0.0 2.30.1 0.0 0.6 0.0 0.2 0.4 2.1 0.0 0.7 1.0 N 0.0 1.6 0.7 0.4 0.3 0.0 0.30.0 0.0 0.4 1.5 0.2 0.7 P 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.2 0.0 0.7 Q 0.00.3 2.1 0.0 0.6 0.2 0.0 0.0 0.0 0.4 1.8 0.0 0.7 R 0.0 1.0 0.3 0.0 0.50.0 0.2 1.2 0.0 0.2 1.2 S 0.0 2.7 0.5 0.4 0.5 0.5 0.0 0.0 0.3 0.0 0.10.9 0.1 T 0.0 1.5 0.5 0.0 0.3 0.4 0.0 0.0 0.0 0.3 1.4 0.0 V 0.0 7.0 1.90.0 0.6 0.0 0.0 0.0 0.6 2.1 0.0 2.8 W 0.0 1.8 6.5 0.0 1.1 0.0 0.0 0.72.2 0.3 0.0 1.4 Y 3.3 0.3 0.0 0.2 0.9 0.1 0.0 0.6 3.2 0.0 1.0 1.5

Of the modifications expected to result in increased kynurenine binding,the sequence into which the modification Lch_Ser56Tyr was added wasnamed F02h011/F02l098 (heavy chain variable region sequence: SEQ ID NO:95; light chain variable region sequence: SEQ ID NO: 97). The sequencewas used as a template sequence for a library. The binding ofF02h011/F02l098 to kynurenine was measured by the Biacore-based methoddescribed in Example (7-5), except that the time of adding the dilutedantigen solutions and the running buffer as a blank was modified to 30seconds and the type of sensor chip immobilized with Protein A waschanged to Sensor chip Series S CM3 (GE Healthcare). Kinetic parametersfor the kynurenine binding of F02h011/F02l098 were determined: ka=3686(l/s), kd=0.26 (l/s), and KD=0.072 (mmol/L). Sensorgrams showing theassessment results are shown in FIG. 33.

(8-3) Assessment of Comprehensive Variants for Library Design Based onResults of X-Ray Crystallographic Structure Analysis

The crystallographic structure of the complex of 6RNMSC1-2_F02 andkynurenine was analyzed in Example (8-1). The mode of recognition bywhich the antibody recognizes kynurenine and the amino acid residues ofthe antibody variable region that are presumed not to be significantlyinvolved in kynurenine binding were identified based on the result ofcrystallographic structure analysis. It was conceived that, bycomprehensively evaluating variants whose residues shown below aresubstituted with each of the amino acids, the sites that can be madeinto a library and the amino acids that can be made into a library couldbe determined. Specifically, it was conceived that, by evaluating thesites that are not greatly involved in the binding toward kynurenine, orthe sites in which amino acids other than those of the native sequencethat may be involved in binding but do not significantly reduce thebinding toward kynurenine (do not render the binding to zero) arepresent, as well as the amino acids, the sites that can be made into alibrary and the amino acids that can be made into a library could bedetermined. Several variants were generated by introducing modificationsto these residues in F02h011/F02l098, which was generated in Example(8-2-4) and in which a modification that increases kynurenine bindinghad been added.

Of the sites in the heavy chain, the modified sites (the sites shownaccording to Kabat numbering and indicated as “Kabat” in the table), theamino acids before modification at these sites (the amino acidsindicated as “native sequence” in the table), and the amino acids aftermodification (the amino acids indicated as “altered amino acids” in thetable) are shown in Table 19.

TABLE 19 FR1 HCDR1 HCDR2 FR3 HCDR3 Kabat 24 26 27 28 29 30 31 33 51 5252a 53 54 55 56 58 73 95 98 99 100a 100b 100e 100f 101 Native sequence AG G T F S S A I I P I F G T N E D V V R P A F D Altered A A A A A A Aamino D D D D D D D D D D D D D D D D D D D D D D D acid E E E E E E E EE E E E E E E E E E E E E E E E F F F F F F F F F F F F F F F F F F F FF F G G G G G G G G G G G G G G G G G G G G G G G H H H H H H H H H H HH H H H H H H H H H H H H H H I I I I I I I I I I I I I I I I I I I I IK K K K K K K K K K K K K K K K K K K K K K K K K K L L L L L L L L L LL L L L L L L L L L L L L L M M M M N N N N N N N N N N N N N N N N N NP P P P P P P P P P P P P P P P P P P P P P P P Q Q Q Q Q Q Q Q Q Q Q QQ Q Q Q Q Q Q Q Q Q Q Q R R R R R R R R R R R R R R R R R R R R R R R RR S S S S S S S S S S S S S S S S S S S S S T T T T T T T T T T T T T TT T T T T T T T V V V V V V V V V V V V V V V V V V V V V V V W W W W WW W W W W W W W W W W W W W W W W W W W W Y Y Y Y Y Y Y Y Y Y Y Y Y Y YY Y Y Y Y Y Y Y Y Y

Of the sites in the light chain, the modified sites (the sites shownaccording to Kabat numbering and indicated as “Kabat” in the table), theamino acids before modification at these sites (the amino acidsindicated as “native sequence” in the table), and the amino acids aftermodification (the amino acids indicated as “altered amino acids” in thetable) are shown in Table 20.

TABLE 20 LCDR1 FR2 LCDR2 LCDR3 Kabat 27d 27e 29 32 46 50 51 52 53 54 5592 93 94 96 Native sequence H S G Y L E I S N R F T Q F R Altered A Aamino D D D D D D D D D D D D D D D D acid E E E E E E E E E E E E E E FF F F F F F F F F F F F G G G G G G G G G G G G G G G H H H H H H H H HH H H H H I I I I I I I I I I I I I I I K K K K K K K K K K K K K K K KL L L L L L L L L L L L L L L M M M M N N N N N N N N N N N N P P P P PP P P P P P P P P P P Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q R R R R R R R R R RR R R R S S S S S S S S S S S S T T T T T T T T T T T T T V V V V V V VV V V V V V V V V W W W W W W W W W W W W W W W W Y Y Y Y Y Y Y Y Y Y YY Y Y

The binding of each variant expressed and purified by the methoddescribed in Reference Example 1 described below to kynurenine wasmeasured by the method using Biacore described in Example (7-5), exceptthat the time of adding the diluted antigen solutions and the runningbuffer as a blank was modified to 30 seconds. Meanwhile, the type ofsensor chip immobilized with Protein A was changed to Sensor chip SeriesS CM3 (GE Healthcare) to measure some of the variants. When kynureninedissociation was too fast to determine the dissociation rate constant, adissociation constant KD (M) was calculated by analysis of equilibriumvalues based on the degree of binding response during the interactionwith each antigen (association phase). In this case also, Biacore T200Evaluation Software (GE Healthcare) was used to calculate theparameters. Based on the assay result, the affinity of each variant forkynurenine was calculated as KD values. The result of comparing the KDvalues of each of the heavy chain variants and F02h011/F02l098, which isthe parental sequence, for kynurenine is shown in Table 21. The resultof comparing the KD values of each of the light chain variants andF02h011/F02l098, which is the parental sequence, for kynurenine is shownin Table 22.

TABLE 21 FR1 HCDR1 HCDR2 Kabat 24 26 27 28 29 30 31 33 51 52 52a 53 5455 56 58 Native sequence A G G T F S S A I I P I F G T N Altered A 4.52.0 0.6 0.0 1.0 amino D 2.1 2.5 4.6 0.7 0.0 0.8 2.6 0.0 0.0 0.1 0.0 0.30.6 1.2 0.7 0.4 acid E 11.0 2.0 6.0 1.1 0.0 1.1 2.4 0.0 0.1 0.4 0.0 0.20.4 0.7 0.7 0.2 F 0.4 2.6 4.9 1.2 1.5 1.1 0.0 0.2 0.3 0.0 0.5 0.3 1.20.7 G 1.4 0.7 0.0 0.4 1.3 0.4 0.4 0.1 0.3 0.8 0.8 0.3 0.8 H 1.5 2.5 3.30.6 0.0 1.2 1.1 0.0 0.1 0.2 0.0 0.5 0.7 1.3 0.8 1.0 I 0.8 4.3 2.6 1.40.3 0.9 0.4 2.5 0.1 0.9 0.3 1.1 0.1 K 1.1 7.9 3.4 0.5 0.0 1.1 1.0 0.30.4 0.7 0.0 0.5 0.8 0.8 0.9 1.1 L 0.9 5.3 4.4 1.1 0.5 2.0 1.3 0.2 0.30.5 0.0 0.8 0.7 0.5 1.6 0.1 M 1.9 1.2 N 2.6 4.8 1.1 0.9 0.0 0.2 0.4 0.00.4 1.8 0.6 P 0.8 11.0 1.8 0.4 0.0 0.5 0.0 0.0 0.0 0.5 0.5 1.1 0.6 0.70.3 Q 1.1 4.0 3.4 0.9 0.0 1.0 1.8 0.2 0.5 0.7 0.0 0.4 0.6 1.2 0.8 1.0 R0.3 9.1 4.2 0.5 0.0 1.1 0.8 0.4 0.7 0.3 0.0 0.5 0.8 0.5 0.9 1.6 S 2.11.6 3.4 1.0 0.0 0.5 0.4 0.3 0.3 0.5 0.8 0.9 0.8 0.7 T 3.6 2.1 3.3 0.01.5 0.5 0.4 0.3 0.6 0.2 0.8 0.9 0.6 1.1 V 1.0 4.9 4.4 0.8 0.2 1.0 0.50.7 1.8 1.0 0.0 1.2 0.8 0.4 0.8 0.6 W 0.5 7.6 9.7 23.6 0.3 1.5 1.2 8.30.2 0.5 0.2 0.3 1.0 1.0 5.1 1.0 Y 2.2 4.6 20.6 0.7 0.5 1.2 0.8 0.0 0.60.3 0.0 0.5 1.1 0.5 0.9 1.5 FR3 HCDR3 Kabat 73 95 98 99 100a 100b 100e100f 101 Native sequence E D V V R P A F D Altered A 1.1 amino D 0.9 0.00.2 0.4 0.0 0.0 acid E 0.0 0.0 0.2 0.3 0.0 0.0 0.0 F 0.8 0.0 3.4 0.1 0.00.0 0.0 G 1.1 1.2 0.0 0.1 0.0 0.1 1.4 0.0 0.0 H 0.9 0.0 0.0 0.8 0.1 0.00.0 0.1 0.0 I 1.1 0.0 1.0 0.5 0.0 0.0 0.0 K 1.1 0.0 0.0 0.4 0.7 1.5 0.00.0 0.0 L 2.0 0.0 0.6 0.4 0.0 2.2 0.0 M 1.6 N 0.0 0.0 0.5 0.3 0.3 0.0 P1.0 0.0 0.0 0.0 1.2 0.0 0.0 0.0 Q 0.9 0.0 0.0 0.4 0.6 0.3 0.0 R 1.3 0.00.2 0.7 0.5 0.0 0.0 0.0 S 1.0 0.0 0.0 0.4 0.4 0.0 T 1.3 0.0 0.5 0.5 0.20.0 0.0 V 1.4 0.0 0.6 0.0 0.0 0.0 W 0.7 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0Y 0.4 0.0 1.9 0.0 0.0 0.0 0.1 0.0

TABLE 22 LCDR1 FR2 LCDR2 LCDR3 Kabat 27d 27e 29 32 46 50 51 52 53 54 5592 93 94 96 Native sequence H S G Y L E I S N R F T Q F R Altered A 0.0amino D 1.9 0.9 1.0 0.0 0.0 0.0 0.4 0.8 1.1 0.8 0.0 1.2 0.9 0.0 0.0 acidE 1.3 0.9 0.9 0.2 0.3 1.0 0.9 0.9 0.0 0.4 1.6 0.0 0.0 F 0.6 0.9 1.0 2.00.5 0.4 1.1 1.0 0.9 0.9 0.5 0.0 G 1.1 0.9 0.3 0.0 0.4 0.4 1.0 0.5 0.90.1 1.4 0.4 0.2 0.0 H 1.0 1.0 0.8 0.0 0.2 0.9 0.9 1.2 0.1 1.0 0.1 0.40.0 I 2.3 0.9 1.2 0.0 0.4 0.4 1.0 1.2 0.8 0.1 2.2 0.6 0.9 0.0 K 1.1 1.01.1 0.5 0.0 0.2 0.3 1.2 1.2 0.9 0.1 1.1 0.6 0.4 0.0 L 1.8 0.9 1.0 0.40.8 0.2 0.9 1.4 0.8 0.2 1.0 0.4 0.2 0.0 M 0.4 1.9 0.4 0.9 N 2.3 1.0 0.30.2 0.8 0.7 0.1 1.8 1.1 0.2 0.0 P 2.3 0.9 1.8 0.4 0.1 0.0 0.0 0.1 0.61.0 0.0 0.1 0.0 0.0 0.0 Q 1.4 1.0 1.0 0.3 0.0 0.8 0.4 1.2 1.0 1.0 0.11.4 0.2 0.0 R 0.9 1.1 0.9 0.9 0.0 0.0 0.5 1.2 1.1 0.1 0.9 0.7 0.3 S 1.00.9 0.0 0.2 0.4 0.8 0.9 0.9 0.8 0.2 0.0 T 2.0 1.0 0.9 0.0 0.3 0.4 1.00.7 0.6 1.7 0.4 0.0 V 2.5 1.0 1.2 0.3 0.6 0.2 0.6 1.1 1.0 0.7 0.1 1.71.0 0.8 0.0 W 0.6 1.0 1.1 0.3 0.0 0.0 0.3 1.0 1.0 0.9 0.0 0.8 0.0 0.80.0 Y 0.6 1.2 1.0 0.2 0.3 1.1 1.1 1.2 1.3 0.9 0.5 1.1 0.0

(8-4) Library Design Based on the Result of Assessment of ComprehensiveVariants

To design a library, sites that meet at least one of the followingconditions were selected as library-constructible sites based on theinformation obtained in Examples (8-2-4) and (8-3) and Example (9-2)described below.

Condition 1: sites that are not greatly involved in the binding towardkynurenine, or sites in which amino acids other than those of the nativesequence are present and which may be involved in binding but which donot significantly reduce the binding toward kynurenine;

Condition 2: sites having a certain level of diversity of amino acidoccurrence frequency as repertoire of the antibody; and

Condition 3: sites that are not important for the formation of canonicalstructures.

From the evaluation results of Examples (8-2-4) and (8-3) and Example(9-2) described below, when sites for which at least one or morevariants, whose KD values toward kynurenine exceed 20% of that of theparent sequence in each evaluation, exist, those were judged to bemodifiable sites that meet the above-described conditions. Of the aminoacids substituted at these sites, the amino acids, whose KD valuestoward kynurenine exceed 20% of that of the parent sequence in eachevaluation were judged to be library-constructible amino acids (flexibleresidues that can be made to appear in the library). A library forobtaining kynurenine switch antibodies is constructed by designing alibrary in which at least any one or more amino acids from among theamino acids contained in the amino acid repertoire, which includes thelibrary-constructible amino acids selected from the variant analysis(the flexible residues that can be made to appear in the library) andthe amino acids of the non-modified antibody (i.e., the amino acidsincluded in the native sequence of F02h011/F02l098), appear at thedetermined modifiable sites in F02h011/F02l098, which was generated inExample (8-2-4) and in which a modification that increases kynureninebinding had been added. The sites containing an amino acid repertoire inthe heavy chain, and the amino acid repertoires at these sites are shownin Table 23. The sites containing an amino acid repertoire in the lightchain, and the amino acid repertoires at these sites are shown in Table24. In the tables, sites shown according to Kabat numbering indicated by“Kabat” represent modifiable sites; amino acids indicated by “nativesequence” represent non-modified amino acids at these sites; and aminoacids indicated by “library-constructible amino acids” representlibrary-constructible amino acids at these sites. A library, in which atleast any one amino acid from among the amino acids contained in theselected amino acid repertoire appears at each of the modifiable sites,was designed.

TABLE 23 FR1 HCDR1 HCDR2 Kabat 24 26 27 28 29 30 31 32 33 50 51 52 52a53 54 55 58 68 Native sequence A G G T F S S Y A G I I P I F G T NLibrary- A A A A A A A A A A A A A A A construct- D D D D D D D D D D DD ible amino E E E E E E E E E E E E E acid F F F F F F F F F F F F F FG G G G G G G G G G G G H H H H H H H H H H H H H H H I I I I I I I I II I I I K K K K K K K K K K K K K K K K L L L L L L L L L L L L L L L MM M M N N N N N N N N N N P P P P P P P P P P P P Q Q Q Q Q Q Q Q Q Q QQ Q Q Q Q R R R R R R R R R R R R R R R R S S S S S S S S S S S S S S ST T T T T T T T T T T T T T T V V V V V V V V V V V V V V V V W W W W WW W W W W W W W W W W W W Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y FR3 HCDR3Kabat 73 95 96 97 98 99 100 100a 100b 100c 100d 100e 100f 102 Nativesequence E D A P V V A R P R G A F I Library- A A A A A A A construct- DD D D D D ible amino E E E E E acid F F F F F F G G G G G H H H H H H HI I I I I I K K K K K K K K L L L L L L L L M M M M N N N N N N N N N PP P Q Q Q Q Q Q Q Q Q R R R R R R R S S S S S S S S S T T T T T T T T VV V V V V W W W W Y Y Y Y Y Y

TABLE 24 LCDR1 FR2 LCDR2 LCDR3 Kabat 27d 27e 28 29 32 46 49 50 51 52 5354 55 92 93 94 Native sequence H S D G Y L Y E I S N R F T Q F Library-A A A A A A A A A A A A A construct- D D D D D D D D D D ible amino E EE E E E E E E E E acid F F F F F F F F F F F F F F G G G G G G G G G G GG H H H H H H H H H H H H I I I I I I I I I I I I I K K K K K K K K K KK K K K L L L L L L L L L L L L L M M M M M M N N N N N N N N N N P P PP P P P P Q Q Q Q Q Q Q Q Q Q Q Q Q R R R R R R R R R R R R R S S S S SS S S S S S T T T T T T T T T T T T V V V V V V V V V V V V V V V W W WW W W W W W W W W W Y Y Y Y Y Y Y Y Y Y Y Y Y Y

Genes containing each of the sequences included in the library designedin this manner are synthesized, and using the collection (library) ofthese individual genes as a template, a gene library is amplified withprimers that can amplify each of VH and VL. The amplified gene libraryof rationally designed human antibody heavy chain variable regions andthat of the human antibody light chain variable regions are introducedinto appropriate phagemid vectors having both a human IgG-derived CH1sequence and a human IgG-derived light chain constant region sequence. Arationally designed library which enables to obtain antibodies whichbind to antigens using kynurenine as a switch are constructed byintroducing these phagemid vectors into Escherichia coli byelectroporation and then presenting Fab domains consisting of a humanantibody variable region-constant region. Such a rationally designedlibrary composed of diverse H chains and L chains having bindingactivities to kynurenine is thought to be useful as a library containinghuman antibodies that enable to efficiently obtain kynurenine switchantibodies against arbitrary antigens. Moreover, since, as described inExample (7-6), 6RNMSC1-2_F02 binds not only to kynurenine but also to3-hydroxykynurenine which is metabolite of kynurenine and kynureninederivatives including RO0635389-000-001, it is also predicted to havebinding activity to other derivatives that are structurally similar tokynurenine. Thus, the library is considered to be useful for obtainingswitch antibodies whose binding activity toward an arbitrary targetantigen varies depending on the presence or absence of any one or moresmall molecules of kynurenine, metabolites of kynurenine, and kynureninederivatives that are exogenous molecules.

Example 9 Library Design for Obtaining Kynurenine-Switch Antibodies byPanning with a Molecule Acting as a Switch

Example 8 describes a method for constructing a library using anantibody having binding activity to kynurenine as a template anddesigning the sites for making a library by comprehensive modification.As a different approach for producing a library, a method using panningwith a molecule that serves as a switch is also useful.

(9-1) X-Ray Crystallographic Analysis of Antibody 6RNMSC1-2_F02 thatBinds to hIL-6R with Kynurenine as a Switch

The crystal structure of a complex of 6RNMSC1-2_F02 and kynurenine wasanalyzed as described in Example (8-1). The mode at which kynurenine isrecognized by the antibody as well as amino acid residues in theantibody variable region presumably not greatly involved in kynureninebinding were identified based on the results of crystallographicanalysis.

(9-2) Assessment for Selecting Library-Constructible Sites, and LibraryDesign

The present inventors conceived that library-constructible sites can beselected through assessment of variants produced by substituting Ala orVal for the various positions selected based on results of thecrystallographic analysis of F02h011/F02l003 (heavy chain variableregion sequence, SEQ ID NO: 95; light chain variable region sequence,SEQ ID NO: 96) generated as described in Example (8-2-2).

(9-2-1) Assessment for Selecting Library-Constructible Sites in theHeavy Chain Variable Region, and Library Design

First, regarding the heavy chain, among the sites contained in the heavychain sequence of F02h011/F02l003, variants were generated bysubstituting Ala or Val for the various positions selected based oncrystallographic analysis results. The modified sites (positionsaccording to Kabat numbering which are indicated as “Kabat” in thetable), amino acids before modification at these sites (amino acidsindicated as “native sequence” in the table), and amino acids aftermodification (amino acids indicated as “altered amino acids” in thetable) are shown in Table 25.

TABLE 25 HFR1 HCDR1 HCDR2 Kabat 28 31 32 33 35 50 51 52 52a 53 54 55 5658 Native sequence T S Y A S G I I P I F G T N Altered A A A V A A A A AA A A A A amino acid HCDR3 Kabat 95 96 97 98 99 100 100a 100b 100c 100d100e 100f 101 102 Native sequence D A P V V A R P R G A F D I Altered AV A A A V A A A A V A A A amino acid

Each variant expressed and purified by the method described in ReferenceExample 1 below was assayed for its kynurenine binding by theBiacore-based method described in Example (7-5), except that the periodof antigen loading was changed to be 30 seconds. When kynureninedissociation was too fast to determine the dissociation rate constant,the dissociation constant KD (M) was calculated by analysis ofequilibrium values based on the degree of binding response when it isinteracted with each antigen (association phase). Also in this case, theBiacore T200 Evaluation Software (GE Healthcare) was used to calculateparameters. Based on the assay results, KD values were calculated todetermine the affinity of each variant for kynurenine. Each variant andits parental antibody F02h011/F02l003 were compared in terms of the KDvalue for kynurenine. The results are shown in Table 26.

TABLE 26 HFR1 HCDR1 HCDR2 Kabat 28 31 32 33 35 50 51 52 52a 53 54 55 5658 Native sequence T S Y A S G I I P I F G T N Altered A A A V A A A A AA A A A A amino acid F02h011/ 0.9 1.7 0.0 0.7 0.7 6.9 0.5 0.4 0.4 0.40.6 0.8 0.8 0.7 F02l003 ratio HCDR3 Kabat 95 96 97 98 99 100 100a 100b100c 100d 100e 100f 101 102 Native sequence D A P V V A R P R G A F D IAltered A V A A A V A A A A V A A A amino acid F02h011/ 0.0 1.9 0.5 0.10.5 0.6 0.3 0.2 0.1 0.2 0.0 0.0 0.0 0.4 F02l003 ratio

Upon designing a library, sites that meet at least one of the followingconditions were selected as library-constructible sites based oninformation obtained in the above assessment of variants and in theassessment described in Example (8-2-4).

Condition 1: sites that are not greatly involved in the binding towardkynurenine, or sites in which amino acids other than those of the nativesequence are present and which may be involved in binding but which donot significantly reduce the binding toward kynurenine (do not renderthe binding to zero);

Condition 2: sites having a certain level of diversity of amino acidoccurrence frequency as repertoire of the antibody; and

Condition 3: sites that are not important for the formation of canonicalstructures.

A library was designed so that the amino acids at positions selected bythe conditions described above appear only at certain nucleotides in theheavy chain sequence (SEQ ID NO: 95) of F02h011/F02l098 produced asdescribed in Example (8-2-4) by introducing a modification that augmentsthe kynurenine binding. Such libraries include, for example, NNK andTRIM libraries (Gonzalez-Munoz A et al., MAbs 2012; Lee C V et al., JMol Biol. 2004; Knappik A. et al., J Mol Biol. 2000; Tiller T et al.,MAbs 2013). Among the various positions assessed, modification sites inwhich a KD value for kynurenine indicates more than 20% binding ofF02h011/F02l003, which is the parental sequence, were judged to bemodifiable positions that meet the above-described conditions. However,even if included in such sites, residue positions judged to bestructurally important were excluded from the sites to be included in alibrary, or were included in a library with selected types of occurringamino acids. Sites for library production using NNK codons (indicated byan open circle in the table), sites fixed to the native sequence(indicated by a cross in the table), and amino acids of selectedoccurring amino acid types are shown in Table 27.

TABLE 27 HFR1 HCDR1 HCDR2 kabat 28 31 32 33 35 50 51 52 52a 53 54 55 5658 Native sequence T S Y A S G I I P I F G T N Library ∘ ∘ x ∘ x ∘ I, F,V, L ∘ x x ∘ G, S ∘ ∘ HCDR3 kabat 95 96 97 98 99 100 100a 100b 100c 100d100e 100f 101 102 Native sequence D A P V V A R P R G A F D I Library x∘ A, P x ∘ ∘ ∘ A, P R, H x x x x x

The designed gene sequence was synthesized using primers containing atthe sites for library production (heavy chain variable region library)NNK codons or codons in which selected amino acids occur; and insertedinto an appropriate phagemid vector carrying human IgG-derived CH1sequence and human IgG-derived light chain constant region sequence incombination with the light chain variable region sequence ofF02h011/F02l098 (parental sequence, SEQ ID NO: 97). A phage-displaylibrary of human antibody heavy chain variable regions capable ofbinding to an antigen via kynurenine as a switch was constructed byintroducing the phagemid vector into E. coli through electroporation.

(9-2-2) Assessment for Selecting Library-Constructible Sites in theLight Chain Variable Region, and Library Design

Next, from the sites comprised in the light chain sequence ofF02h011/F02l003 in the light chain, various modified variants weregenerated by substituting Ala or Val for the various positions selectedbased on the crystallographic analysis results. The modified sites(positions according to Kabat numbering which are indicated as “Kabat”in the table), amino acids before modification at the sites (amino acidsindicated as “native sequence” in the table), and amino acids aftermodification (amino acids indicated as “altered amino acids” in thetable) are shown in Table 28.

TABLE 28 LCDR1 LCDR2 LCDR3 Kabat 27d 27e 28 29 30 32 52 53 54 55 56 9192 93 94 96 Native sequence H S D G N Y S N R F S A T Q F R Altered A AA A A A A A A A A V A A A A amino acid

Each variant expressed and purified by the method described in ReferenceExample 1 below was assayed for its kynurenine binding by theBiacore-based method described in Example (7-5), except that the periodof antigen loading was changed to 30 seconds. When kynureninedissociation was too fast to determine the dissociation rate constant,the dissociation constant KD (M) was calculated by analyzing equilibriumvalues based on the degree of binding response when it is interactedwith each antigen (association phase). Also in this case, the BiacoreT200 Evaluation Software (GE Healthcare) was used to calculateparameters. Based on the assay results, KD values were calculated todetermine the affinity of each variant for kynurenine. Each variant andits parental sequence F02h011/F02l003 were compared in terms of the KDvalue for kynurenine. The results are shown in Table 29.

TABLE 29 LCDR1 LCDR2 LCDR3 Kabat 27d 27e 28 28 30 32 52 53 54 55 56 9192 93 94 96 Native sequence H S D G N Y S N R F S A T Q F R Altered A AA A A A A A A A A V A A A A amino acid F02h011/ 0.7 0.9 2.5 1.0 0.7 0.51.0 0.8 0.8 0.1 0.9 0.3 0.9 1.0 0.1 0.0 F02l003 ratio

Upon designing a library, sites that meet at least one of the followingconditions were selected as library-constructible sites based on theinformation obtained in the above assessment of modified variants and inthe assessment described in Example (8-2-4).

Condition 1: sites that are not greatly involved in the binding towardkynurenine, or sites in which amino acids other than those of the nativesequence are present and which may be involved in binding but which donot significantly reduce the binding toward kynurenine (do not renderthe binding to zero);

Condition 2: sites having a certain level of diversity of amino acidoccurrence frequency as repertoire of the antibody; and

Condition 3: sites that are not important for the formation of canonicalstructures.

A library was designed so that amino acids at positions selected by theconditions described above appear only at certain nucleotides in thelight chain sequence (SEQ ID NO: 97) of F02h011/F02l098 produced asdescribed in Example (8-2-4) by introducing a modification that augmentsthe kynurenine binding. Such libraries include, for example, NNK andTRIM libraries. Among the various positions assessed, modification sitesin which a KD value for kynurenine indicates more than 20% binding ofF02h011/F02l003, which is the parental sequence, were judged to bemodifiable positions that meet the above-described conditions. However,even if included in such sites, residue positions judged to bestructurally important were excluded from the sites included in alibrary or were included in a library with selected types of occurringamino acids. Sites for library production using NNK codons (indicated byan open circle in the table), sites fixed to the native sequence(indicated by a cross in the table), and amino acids of selectedoccurring amino acid types are shown in Table 30.

TABLE 30 LCDR1 LCDR2 LCDR3 Kabat 27d 27e 28 29 30 32 52 53 54 55 56 9192 93 94 96 Native sequence H S D G N Y S N R F S A T Q F R Library ∘ ∘∘ ∘ x Y, F ∘ ∘ ∘ x Y x ∘ ∘ x x

The designed gene sequence was synthesized using primers containing atthe sites for library production (light chain variable region library)NNK codons or codons in which selected amino acids occur; and insertedinto an appropriate phagemid vector carrying human IgG-derived CH1sequence and human IgG-derived light chain constant region sequence incombination with the heavy chain variable region sequence ofF02h011/F02l098 (parental sequence, SEQ ID NO: 95). A phage-displaylibrary of human antibody light chain variable regions capable ofbinding to an antigen via kynurenine as a switch was constructed byintroducing the phagemid vector into E. coli through electroporation.

(9-3) Synthesis of Biotinylated Kynurenine to Construct aKynurenine-Binding Library

As shown in Example (7-6), kynurenine derivatives also function as aswitch for 6RNMSC1-2_F02. In their structure, the derivatives have asubstitution at any of the positions 3, 4, and 5 of kynurenine. Further,the result of crystallographic analysis on the complex of 6RNMSC1-2_F02and kynurenine allows prediction that biotin introduced at position 3,4, or 5 of kynurenine does not impede the binding of 6RNMSC1-2_F02 tothe antigen hIL-6R. Thus, it is preferable that an appropriate linker(such as PEG linker) is linked at position 3, 4, or 5, and biotin isadded to the end of the linker.

An example of biotinylated kynurenine of the present invention isdescribed below. Biotinylated kynurenine of the present invention can besynthesized by various methods. Some of them are illustrated using theschemes below. The schemes are an example, and the present invention isnot limited to the chemical reactions and conditions shown herein. Therepresentative compounds of the present invention can be synthesizedusing appropriate intermediates, known compounds, and reagents.

(9-3-1) Synthesis of Biotinylated Compounds in which a Linker is Linkedto Position 4 of Kynurenine

Biotinylated kynurenine compounds 028 and 029 can be prepared by themethod described below. The biotinylated kynurenine compounds andsynthetic intermediates were analyzed under the conditions describedbelow.

The condition of LCMS analysis is as follows.

TABLE 31 Flow Column Analysis Column (particle diameter, ratetemperature condition Apparatus length, inner diameter) Mobile phaseGradient (A/B) (ml/min) (° C.) Wavelength A Shimadzu Shim-pack XR-ODS A:Water/0.05% TFA 95/5 (0.01 min), 1 40 190-800 nm, LCMS-2020 (2.2 um, 50mm, 3.0 mm) B: Acetonitrile/0.05% TFA 0/100 (1.20 min), PDA total 0/100(2.20 min), 95/5 (2.30 min) B Shimadzu Shim-pack XR-ODS A: Water/0.05%TFA 95/5 (0.01 min), 1 40 190-800 nm, LCMS-2020 (2.2 um, 50 mm, 3.0 mm)B: Acetonitrile/0.05% TFA 0/100 (2.20 min), PDA total 0/100 (3.20 min),95/5 (3.30 min) C Shimadzu ACQUITY UPLC BEH C18 A: Water/0.05% TFA 95/5(0.01 min), 1 45 190-800 nm, LCMS-2020 (1.7 um, 50 mm, 2.1 mm) B:Acetonitrile/0.05% TFA 0/100 (1.20 min), PDA total 0/100 (1.90 min),95/5 (2.00 min) D Shimadzu Shim-pack XR-ODS A: Water/0.05% TFA 95/5(0.01 min), 1 40 190-800 nm, LCMS-2020 (2.2 um, 50 mm, 3.0 mm) B:Acetonitrile/0.05% TFA 35/65 (4.50 min), PDA total 35/65 (5.60 min),95/5 (5.70 min) E Shimadzu ACQUITY UPLC BEH C18 A: Water/0.05% TFA 95/5(0.01 min), 1 45 190-800 nm, LCMS-2020 (1.7 um, 50 mm, 2.1 mm) B:Acetonitrile/0.05% TFA 0/100 (1.00 min), PDA total 0/100 (1.80 min),95/5 (1.90 min) F Shimadzu Ascentis Express C18 A: water/0.1% FA 90/10(0.01 min), 1.5 40 190-800 nm, LCMS-2020 (2.7 um, 50 mm, 3.0 mm) B:Acetonitrile/0.05% FA 0/100 (1.10 min), PDA total 0/100 (1.60 min),90/10 (1.70 min) G Shimadzu Phenomenex kinetex A: water/0.1% FA 90/10(0.01 min), 1.5 40 190-800 nm, LCMS-2020 (2.6 um, 50 mm, 3.0 mm) B:Acetonitrile/0.05% FA 0/100 (2.00 min), PDA total 0/100 (2.70 min),90/10 (2.80 min) H Aquity Ultra Ascentis Express C18 A: water/0.1% FA60/40 (0.01 min), 1 25 190-800 nm, Performance (2.7 um, 50 mm, 3.0 mm)B: Acetonitrile/0.05% FA 0/100 (1.40 min), PDA total 0/100 (1.40 min),60/40 (3.0 min)

Sodium bis(trimethylsilyl)amide (38% tetrahydrofuran solution, 1.9mol/l, 21.6 ml, 41.1 mmol) was added over 15 minutes to atetrahydrofuran solution (100 ml) containing 5-bromo-2-iodoaniline(compound 019, 4.90 g, 16.5 mmol, COMBI-BLOCKS) and di-t-butyldicarbonate (7.54 g, 41.1 mmol) at 0° C. in a nitrogen gas flow. Afterstirring at 0° C. for 30 minutes and then at room temperature for 18hours, the reaction solution was diluted with ethyl acetate (300 ml).Following wash with 1 M hydrochloric acid, a saturated aqueous solutionof sodium bicarbonate and a saturated saline solution, the mixture wasdried over sodium sulfate and concentrated under reduced pressure. Theresulting residue was purified by normal phase silica gel columnchromatography (ethyl acetate/hexane). This yielded compound 020 (6.14g, 90%).

LCMS (ESI) m/z=342, 344 (M−Bu+H)+

Retention time: 0.97 minute (analysis condition H)

Methylmagnesium bromide (1 M, 33.2 ml, 33.2 mmol) was added to atetrahydrofuran solution (120 ml) of N-t-butoxy-(5-bromo-2-iodophenyl)carbamate (compound 020, 12.0 g, 30.2 mmol) at −78° C. in a nitrogen gasflow, and the mixture was stirred at the same temperature for one hour.Then, after adding n-butyllithium (2.5 M, 13.3 ml, 33.3 mmol), themixture was stirred at −78° C. for two hours. A tetrahydrofuran solution(15 ml) of (2S)-2-[[t-butoxycarbonyl]amino]-4-oxobutanoate (compound021, 4.20 g, 15.4 mmol; Roberts et al., Bioorg. Med. Chem. Letters, 13(2), 265-267 (2003)) was added to the above mixture at −78° C. over 30minutes, and the mixture was further stirred at the same temperature fortwo hours. 1 M hydrochloric acid was added to the reaction solution. Thereaction solution was extracted three times with ethyl acetate. Afterwashing with 1 M hydrochloric acid, a saturated aqueous solution ofsodium bicarbonate and a saturated saline solution, the collectedorganic layer was concentrated under reduced pressure. The resultingresidue was purified by normal phase silica gel column chromatography(ethyl acetate/petroleum ether). This yielded compound 022 (7.50 g,89%).

LCMS (ESI) m/z=569 (M+Na)+

Retention time: 1.93 minute (analysis condition A)

Dess-Martin Periodinane (11.7 g, 27.6 mmol) was added to adichloromethane solution (100 ml) of compound 022 (7.50 g, 13.8 mmol) at25° C., and the mixture was stirred for two hours. After diluting withethyl acetate, the reaction mixture was washed with an aqueous sodiumthiosulfate solution, an aqueous sodium hydrogen carbonate solution, anda saturated saline solution, and concentrated under reduced pressure.The resulting residue was purified by normal phase silica gel columnchromatography (ethyl acetate/petroleum ether). This yielded compound023 (5.80 g, 78%).

LCMS (ESI) m/z=565 (M+Na)+

Retention time: 1.78 minute (analysis condition A)

A dioxane solution (60 ml) containing compound 023 (2.50 g, 4.58 mmol),copper(I) iodide (438 mg, 2.30 mmol),(1R,2R)-(−)-N,N′-dimethylcyclohexane-1,2-diamine (654 mg, 4.60 mmol),and sodium iodide (1.40 g, 9.34 mmol) was stirred at 120° C. undernitrogen atmosphere for 16 hours. The residue obtained by concentratingthe reaction mixture under reduced pressure was purified by normal phasesilica gel column chromatography (ethyl acetate/petroleum ether). Thisyielded compound 024 (1.60 g, 59%).

LCMS (ESI) m/z=613 (M+Na)+

Retention time: 1.95 minute (analysis condition B)

A tetrahydrofuran solution (20 ml) containing compound 024 (1.60 g, 2.71mmol), tetrakis(triphenylphosphine) palladium (627 mg, 0.54 mmol),copper iodide (103 mg, 0.54 mmol), bis(2-propynyl)ether (893 mg, 9.49mmol), and triathylamine (960 mg, 9.49 mmol) was stirred at 25° C. undernitrogen atmosphere for 16 hours. The residue obtained by concentratingthe reaction mixture under reduced pressure was purified by normal phasesilica gel column chromatography (ethyl acetate/petroleum ether). Thisyielded compound 025 (700 mg, 46%).

LCMS (ESI) m/z=579 (M+Na)+

Retention time: 2.68 minutes (analysis condition B)

A mixed solution of t-butanol (2.0 ml) and tetrahydrofuran (2.0 ml)containing compound 025 (60.0 mg, 0.11 mmol), biotin-PEG4-N3 (compound026, 60.0 mg, 0.13 mmol), copper sulfate pentahydrate (10.0 mg, 0.040mmol), and an aqueous solution of 1 M sodium ascorbate (five drops) wasstirred at 25° C. for 16 hours. The residue obtained by concentratingthe reaction mixture under reduced pressure was purified by normal phasesilica gel column chromatography (methanol/dichloromethane). Thisyielded compound 027 (100 mg, 91%).

LCMS (ESI) m/z=1001 (M+H)+

Retention time: 1.40 minute (analysis condition C)

Trifluoroacetic acid (2.0 ml) was added to a dichloromethane solution(10 ml) of compound 027 (100 mg, 0.10 mmol), and the mixture was stirredat 25° C. for two hours. The residue obtained by concentrating thereaction mixture under reduced pressure was purified by high-performanceliquid chromatography. This yielded compound 028 (27.8 mg, 37%).

LCMS (ESI) m/z=745 (M+H)+

Retention time: 2.14 minutes (analysis condition D)

Hydrogen gas was flowed over five hours into a methanol solution (10 ml)containing compound 027 (100 mg, 0.10 mmol) and palladium/carbon (20mg). After filtration, the reaction mixture was concentrated underreduced pressure. Trifluoroacetic acid (1.0 ml) was added to adichloromethane solution (10 ml) of the residue. The mixture was stirredat 25° C. for one hour. The residue obtained by concentrating thereaction mixture under reduced pressure was purified by high-performanceliquid chromatography. This yielded compound 029 (13.5 mg, 18%).

LCMS (ESI) m/z=749 (M+H)+

Retention time: 1.20 minute (analysis condition B)

(9-3-2) Synthesis of Biotinylated Compounds in which a Linker is Linkedto Position 5 of Kynurenine

Biotinylated kynurenine compound 036 can be prepared by the methoddescribed below.

Methylmagnesium bromide (1 M, 20 ml, 20 mmol) was added to atetrahydrofuran solution (80 ml) of N-t-butoxy-(4-bromo-2-iodophenyl)carbamate (Wensbo et al., Tetrahedron, 51 (37), 10323-10342 (1995))(compound 030, 7.50 g, 18.8 mmol) at −78° C. in a nitrogen gas flow. Themixture was stirred at the same temperature for 20 minutes. Then, afteradding n-butyllithium (2.5 M, 10 ml, 25 mmol), the mixture was stirredat −78° C. for 30 minutes. To this solution, a tetrahydrofuran solution(20 ml) of (2S)-2-[[t-butoxycarbonyl] amino]-4-oxobutanoate (compound021, 2.57 g, 9.40 mmol) was added over 30 minutes at −78° C. The mixturewas further stirred for one hour at the same temperature. After adding 1M hydrochloric acid, the reaction solution was extracted three timeswith ethyl acetate. The collected organic layer was washed with 1 Mhydrochloric acid, a saturated aqueous solution of sodium bicarbonate,and a saturated saline solution, and dried over sodium sulfate. Theresidue obtained by concentrating under reduced pressure was purified bynormal phase silica gel column chromatography (ethyl acetate/petroleumether). This yielded compound 031 (4.56 g, 89%).

LCMS (ESI) m/z=569 (M+Na)+

Retention time: 1.78 minute (analysis condition A)

Dess-Martin Periodinane (8.89 g, 21.0 mmol) was added to adichloromethane solution (100 ml) of compound 031 (4.56 g, 8.36 mmol) at25° C. The mixture was stirred for three hours. After adding an aqueoussodium thiosulfate solution, the reaction mixture was extracted threetimes with dichloromethane. The combined organic layer was washed withan aqueous sodium hydrogen carbonate solution, and dried over sodiumsulfate. The residue obtained by concentrating under reduced pressurewas purified by normal phase silica gel column chromatography (ethylacetate/petroleum ether). This yielded compound 032 (3.60 g, 79%).

LCMS (ESI) m/z=567 (M+H)+

Retention time: 1.90 minute (analysis condition E)

A dioxane solution (60 ml) containing compound 032 (3.60 g, 6.62 mmol),copper(I) iodide (650 mg, 3.41 mmol),(1R,2R)-(−)-N,N′-dimethylcyclohexane-1,2-diamine (940 mg, 6.61 mmol),and sodium iodide (1.98 g, 13.2 mmol) was stirred at 120° C. undernitrogen atmosphere for 16 hours. The residue obtained by concentratingthe reaction mixture under reduced pressure was purified by normal phasesilica gel column chromatography (ethyl acetate/petroleum ether). Thisyielded compound 033 (3.10 g, 79%).

LCMS (ESI) m/z=613 (M+Na)+

Retention time: 1.33 minute (analysis condition E)

A tetrahydrofuran solution (20 ml) of compound 033 (3.10 g, 5.25 mmol),tetrakis(triphenylphosphine) palladium (1.21 g, 1.05 mmol), copperiodide (200 mg, 1.05 mmol), bis(2-propynyl)ether (1.73 mg, 18.4 mmol),and triathylamine (1.86 g, 18.4 mmol) was stirred at 25° C. undernitrogen atmosphere for 16 hours. The residue obtained by concentratingthe reaction mixture under reduced pressure was purified by normal phasesilica gel column chromatography (ethyl acetate/petroleum ether). Thisyielded compound 034 (1.00 g, 34%).

LCMS (ESI) m/z=579 (M+Na)+

Retention time: 1.28 minute (analysis condition E)

A mixed solution of t-butanol (2 ml) and tetrahydrofuran (2 ml)containing compound 034 (90.0 mg, 0.20 mmol), biotin-PEG4-N3 (compounds026, 90.0 mg, 0.16 mmol), copper sulfate pentahydrate (10 mg, 0.040mmol), and an aqueous solution of 1 M sodium ascorbate (10 drops) wasstirred at 25° C. for 16 hours. The residue obtained by concentratingthe reaction mixture under reduced pressure was purified by normal phasesilica gel column chromatography (methanol/dichloromethane). Thisyielded compound 035 (155 mg, 76%).

LCMS (ESI) m/z=1001 (M+H)+

Retention time: 1.13 minute (analysis condition F)

Hydrogen gas was flowed over four hours into a methanol solution (10 ml)containing compound 035 (85.0 mg, 0.080 mmol) and palladium/carbon (10mg). After filtration, the reaction mixture was concentrated underreduced pressure, and trifluoroacetic acid (1.0 ml) was added to adichloromethane solution (5.0 ml) of the resulting residue. The mixturewas stirred at 25° C. for one hour. The residue obtained byconcentrating the reaction mixture under reduced pressure was purifiedby high-performance liquid chromatography. This yielded compound 036(18.9 mg, 31.6%).

LCMS (ESI) m/z=749 (M+H)+

Retention time: 0.70 minute (analysis condition G)

(9-4) Obtaining a Group of Kynurenine-Binding Antibodies UsingPhage-Display Libraries of Heavy Chain and Light Chain Variable Regions

With biotinylated kynurenine synthesized as described in Example (9-3),panning was carried out to obtain a group of antibodies thatspecifically bind to kynurenine from the respective phage-displaylibraries of the heavy chain and light chain variable regions designedand constructed as described in Examples (9-2-1) and (9-2-2). For thepurpose described above, first, phages presenting antibodies that havebinding activity to magnetic beads in the absence of biotinylatedkynurenine were removed by contacting the antibody-phage-display librarywith magnetic beads in the absence of biotinylated kynurenine. Then, inthe presence of biotinylated kynurenine, panning was performed in thesame manner to screen for antibodies that have specific binding activityto biotinylated kynurenine.

E. coli containing the constructed phage-display phagemid vector wasinfected with M13KO7ΔpIII (PROGEN Biotechnik; referred to ashyperphage), cultured at 30° C. overnight, and phages were collectedfrom the supernatant. To precipitate the group of phages, 2.5 M NaCl/10%PEG was added to the E. coli culture in which phages were produced. Thegroup of phages was diluted with TBS to prepare a solution of anantibody multivalent display phage library. Then, BSA was added at afinal concentration of 4% to the phage library suspension. Panning wasperformed with the antigen immobilized on magnetic beads. The magneticbeads used were NeutrAvidin coated beads (Sera-Mag SpeedBeadsNeutrAvidin-coated) or Streptavidin coated beads (Dynabeads MyOneStreptAvidin T1).

Panning was carried out using a negative selection method to collectphages that specifically bind to kynurenine. Panning was performedindividually for each of the three types of biotinylated kynurenine(compounds 028, 029, and 036) synthesized as described in Example (9-3).Specifically, 0.8 mL of the prepared phage library suspension was addedto BSA-blocked magnetic beads (Dynabeads MyOne StreptAvidin T1), andallowed to bind for 30 minutes at room temperature and then for another30 minutes at 4° C. The beads were separated using a magnetic stand tocollect phages that did not bind to the beads. This process wasrepeated. Separately prepared BSA-blocked magnetic beads (DynabeadsMyOne StreptAvidin T1) were allowed to react with 6 nmol of biotinylatedkynurenine at room temperature for 30 minutes to immobilize biotinylatedkynurenine onto the magnetic beads. The biotinylatedkynurenine-immobilized magnetic beads were washed three times with TBST,and the collected phages were added thereto. The phage library wascontacted with biotinylated kynurenine for 30 minutes at roomtemperature and then for another 30 minutes at 4° C. The beads werewashed twice with 1 mL of ice-cold TBST, and then once with ice-coldTBS. Then, 0.5 mL of TBS containing trypsin at a final concentration of1 mg/mL was added to the beads. Immediately after the beads weresuspended at room temperature for 15 minutes, the phage suspension wascollected from the beads that were separated using a magnetic stand.Phages eluted with the trypsin solution were added to 60 mL of E. colistrain ER2738 in the logarithmic growth phase (OD600=0.4-0.7). The E.coli was gently stirred and incubated at 37° C. for one hour to allowphages to infect the E. coli. The infected E. coli was seeded on threeplates of 225 mm×225 mm. Then, E. coli in the seeded culture wasinfected with hyperphage, and cultured at 30° C. overnight to collect asolution of an antibody multivalent display phage library.

Using the negative selection method in the same way as in the firstpanning, the second panning was carried out individually for each of thethree types of biotinylated kynurenine to collect phages thatspecifically bind to kynurenine. Specifically, first, magnetic beads(Sera-Mag SpeedBeads NeutrAvidin-coated) were blocked with 2% skimmilk-TBS added with 5 μL of 5 mg/mL StreptAvidin recombinant (Roche).The magnetic beads were washed three times with TBST, to which 0.8 mL ofa phage library suspension blocked with 4% BSA was added, and allowed tobind at room temperature for 30 minutes. The beads were separated usinga magnetic stand to collect phages that did not bind to the beads. Thisprocess was repeated. Separately prepared magnetic beads (Sera-MagSpeedBeads NeutrAvidin-coated) blocked with 2% skim milk-TBS added with5 μL of 5 mg/mL StreptAvidin recombinant (Roche) were allowed to reactwith 10 nmol biotinylated kynurenine for 30 minutes at room temperatureand then for another 30 minutes at 4° C., to immobilize biotinylatedkynurenine onto the magnetic beads. The biotinylatedkynurenine-immobilized magnetic beads were washed three times with TBST,and the collected phages were added thereto. The phage library wascontacted with biotinylated kynurenine for 30 minutes at roomtemperature and then for another 30 minutes at 4° C. The beads werewashed three times with 1 mL of ice-cold TBST and twice with ice-coldTBS. Then, 0.5 mL of TBS containing trypsin at a final concentration of1 mg/mL was added to the beads, and immediately after the beads weresuspended at room temperature for 15 minutes, the phage suspension wascollected from the beads that were separated using a magnetic stand. Of0.5 mL of phages eluted with the trypsin solution, 50 μL was added to 60mL of E. coli strain ER2738 in the logarithmic growth phase(OD600=0.4-0.7). The E. coli was gently stirred and incubated at 37° C.for one hour to allow phages to infect the E. coli. The infected E. coliwas seeded on three plates of 225 mm×225 mm.

(9-4-2) Assessment of the Binding Activity for Biotinylated Kynurenineby Phage ELISA

From a single colony of E. coli obtained as described in Example(9-4-1), a phage-containing culture supernatant was collected accordingto a conventional method (Methods Mol. Biol. (2002) 178, 133-145).Antibody multivalent display phages were collected using hyperphages ashelper phages, and subjected to ELISA. A StreptaWell 96 microtiter plate(Roche) was coated with 80 μL of TBS containing three types ofbiotinylated kynurenine (a mixture containing an equal amount ofcompounds 028, 029, and 036) for one or more hours. After biotinylatedkynurenine not bound to StreptAvidin was removed by washing each well ofthe plate with TBST, the wells were blocked with 250 μL of 2% skimmilk-TBS for one or more hours. After removal of 2% skim milk-TBS,purified phages were added to each well. The plate was allowed to standat room temperature for one hour to allow antibody-presenting phages tobind to biotinylated kynurenine in each well. After each well was washedwith TBST, an HRP-conjugated anti-M13 antibody (Amersham PharmaciaBiotech) diluted with TBS was added to the wells. The plate wasincubated for one hour. After TBST washes, the chromogenic reaction ofthe solution in each well added with the TMB single solution (ZYMED) wasterminated by adding sulfuric acid. Then, color development was measuredby absorbance at 450 nm. As a result, several antibodies were confirmedto bind to biotinylated kynurenine. The results of phage ELISA are shownin Table 32.

TABLE 32 Library Heavy chain library Light chain library Panning probeCompound Compound Compound Compound Compound Compound 028 029 036 028029 036 Number of panning rounds 2 2 2 2 2 2 Number of clones 96 96 9696 96 96 subjected to ELISA Number of positive clones 23 17 33 30 17 63(Absorbance > 0.2) Number of positive clone 23 17 33 30 17 63 sequences

(9-4-3) Sequence Analysis of Antibodies that Bind to BiotinylatedKynurenine

Using specific primers (SEQ ID NOs: 79 and 80), genes were amplifiedfrom clones that are judged to have specific binding activity tokynurenine based on the results of phage ELISA shown in Example (9-4-2).The nucleotide sequences of the genes were analyzed, and the analysisresult showed that the sequences of the clones judged to havekynurenine-binding activity were independent of one another.

(9-5) Construction of a Library for Obtaining Kynurenine-SwitchAntibodies by Panning with Kynurenine

Since antigen (kynurenine)-binding phages obtained from each of theheavy chain and light chain variable region phage-display libraries area population having kynurenine-binding ability, a Fab-presenting phagelibrary constructed by combining the two was expected to contain a largenumber of clones that maintain the binding to kynurenine as a switch,implying the possibility of constructing a library that allows moreefficient isolation of kynurenine-switch antibodies.

Using a method known to those skilled in the art, genes were extractedfrom E. coli infected with each of the heavy-chain and light-chain phagelibraries obtained as described in Example (9-4). For the heavy chain, agene library was amplified with primers (SEQ ID NOs: 98 and 99) capableof amplifying the heavy chain variable region using a collection of theobtained genes (heavy chain variable region library) as a template.Regarding the light chain, the heavy chain variable region sequence(library template sequence: SEQ ID NO: 95) of F02h011/F02l098 wasexcised from a collection of the obtained genes (light chain variableregion library) by restriction enzyme treatment to construct a phagemidvector carrying the light chain variable region library gene, humanIgG-derived light chain constant region sequence, and human IgG-derivedCH1 sequence. The heavy-chain variable region library gene was insertedinto the constructed phagemid vector carrying the light chain variableregion library gene to construct a phagemid vector introduced with thehuman antibody heavy chain/light chain variable region library genes. Adesigned library that displays a Fab domain composed of the humanantibody variable region-constant region, which allows isolation ofantibodies that can bind to an antigen via kynurenine as a switch wasconstructed by introducing the phagemid vector into E. coli byelectroporation. Such a designed library composed of various H chainsand L chains having kynurenine-binding activity is expected to be usefulas a library containing human antibodies that allow efficient isolationof kynurenine-switch antibodies against an arbitrary antigen.Furthermore, as shown in Example (7-6), since 6RNMSC1-2_F02 binds notonly to kynurenine, but also to metabolic products thereof:3-hydroxykynurenine and 3-hydroxy-DL-kynurenine, and to kynureninederivatives such as RO0635389-000-001 and RO0635390-000-001, the librarydescribed above is expected to be useful for isolating switch antibodieswhose binding activity to an arbitrary target antigen varies dependingon the presence of any one or more small molecules of kynurenine,metabolic products of kynurenine, and kynurenine derivatives.

Example (9-6) Assessment of the Clones in the Library for theirKynurenine-Binding Ability by Phage ELISA

Since the library constructed as described in Example (9-5) was obtainedvia panning with kynurenine, the library was predicted to contain alarge number of clones having kynurenine-binding ability. Phage clonesisolated from the constructed library were assessed for their binding tobiotinylated kynurenine by phage ELISA.

Phages were cultured according to a conventional method (Methods Mol.Biol. (2002) 178, 133-145) from a single colony of E. coli containinggenes of the constructed library. Culture supernatants containing theantibody multivalent display phages were collected using hyperphages ashelper phages. The collected phage culture supernatants were filtered byultrafiltration using NucleoFast96 (MACHERY-NAGEL). 200 μL each of theculture supernatants was applied to each of the wells of NucleoFast96,and centrifuged at 6000 g for 45 minutes. The flow-through fractionswere removed. Washing was performed by adding 200 μL of H₂O to thewells, and centrifugation at 6000 g for 30 minutes. 200 μL of TBS wasthen added, and after being left to stand at room temperature for fiveminutes, phage suspensions contained in the supernatants were collected.

After TBS addition, the purified phages were subjected to ELISA by thesame method as described in Example (9-4-1). As a result, severalantibodies were confirmed to bind to biotinylated kynurenine. Among theanalyzed phage clones derived from the primary library, about 49% werefound to have kynurenine-binding ability. The results of phage ELISA areshown in Table 33.

TABLE 33 Number of clones subjected to ELISA 192 Number of positiveclones(Absorbance >0.2) 94 Percentage of positive clones 48.96%

Example 10 Assessment of Proteins that Bind to an Antigen Via a SmallMolecule of Non-Biological Origin as a Switch

Antibodies (Reference Example 3 described below) that are capable ofbinding to human IL-6 receptor (hIL-6R) in the presence of ATP oradenosine, which were obtained from a library constructed based onantibody ATNLSA1-4_D12 that binds to ATP and adenosine, were assessedfor their human IL-6 receptor-binding ability in the presence of a smallmolecule of non-biological origin.

(10-1) Preparation of ATP/Adenosine-Switch Antibodies Obtained from aLibrary

The heavy chain and light chain variable region sequences of clones6RAD2C1-4_001, 6RAD2C1-4_005, 6RAD2C1-4_011, 6RAD2C1-4_026,6RAD2C1-4_030, 6RAD2C1-4_042, 6RAD2C1-4_076, 6RDL3C1-4_085, and6RDL3C5-4_011 which were obtained as described in Reference Example 3below and judged to have binding activity to biotin-labeled hIL-6R inthe presence of ATP or adenosine, were each inserted into a humanIgG1/Lambda plasmid for animal expression carrying the antibody heavychain constant region (SEQ ID NO: 46) or light chain Lambda constantregion sequence (SEQ ID NO: 100). The antibodies were expressed usingthe method described below. Cells of the FreeStyle 293-F line(Invitrogen), which is derived from human embryonic kidney cells, weresuspended at a cell density of 1.33×10⁶ cells/mL in the FreeStyle 293Expression Medium (Invitrogen), and 3-mL of the suspension was seeded ineach well of 6-well plates. The prepared plasmid was introduced into thecells by lipofection. After four days of culture in a CO₂ incubator (37°C., 8% CO₂, 90 rpm), antibodies were purified from culture supernatantsby a method known to those skilled in the art using rProtein ASepharose™ Fast Flow (Amersham Biosciences). Absorbance of solutions ofthe purified antibodies was measured at 280 nm using aspectrophotometer. From the values obtained by measurement,concentrations of the purified antibodies were calculated using anextinction coefficient determined by the PACE method (Protein Science(1995) 4, 2411-2423).

(10-2) Assessment of Various Small Molecules for their Effect on theBinding to Human IL-6 Receptor by Surface Plasmon Resonance

Biacore T200 (GE Healthcare) was used to assess various small moleculesfor their effect on the antigen-antibody reaction between hIL-6R andnine clones of ATP/adenosine-dependent antibody (6RAD2C1-4_001,6RAD2C1-4_005, 6RAD2C1-4_011, 6RAD2C1-4_026, 6RAD2C1-4_030,6RAD2C1-4_042, 6RAD2C1-4_076, 6RDL3C1-4_085, and 6RDL3C5-4_011) isolatedfrom a library. The running buffer used was 20 mmol/L ACES, 150 mmol/LNaCl, 0.05% (w/v) Tween20, pH 7.4. The assay was performed at 25° C.hIL-6R was immobilized onto Sensor chip CM5 by amine coupling, andinteracted with the antibodies as an analyte for 120 seconds to observechanges in the amount of binding. Antibodies were diluted with therunning buffer or the running buffer added with any one of ATP, ADP,AMP, cAMP, and adenosine (ADO). The final concentration of each smallmolecule was adjusted to 1 mM, and the final concentration of theantibody was adjusted to 1 μM. Meanwhile, under the 1 mM ATP condition,the assay was carried out with a series of stepwise antibodyconcentrations. The dissociation constant KD (mol/L) of each clone forhIL-6R was calculated from a plot of equilibrium values against antibodyconcentration. The parameters were calculated using the Biacore T200Evaluation Software (GE Healthcare). The dissociation constant KD ofeach clone in the presence of 1 mM ATP is shown in Table 34.

TABLE 34 Dissociation constant K_(D) Clone name (mol/L) 6RAD2C1-4_013.0E−07 6RAD2C1-4_05 3.4E−07 6RAD2C1-4_11 2.3E−07 6RAD2C1-4_26 2.1E−076RAD2C1-4_30 3.3E−07 6RAD2C1-4_42 2.5E−07 6RAD2C1-4_76 2.5E−076RDL3C1-4_85 3.9E−07 6RDL3C5-4_11 1.3E−07

The amount of each clone binding to hIL-6R in the absence or presence ofeach small molecule at 1 mM obtained by this measurement is shown inFIG. 37. As shown in FIG. 37, each clone was bound to hIL-6R in thepresence of 1 mM ATP, while binding to hIL-6R was not observed in theabsence of ATP. Thus, the clones were confirmed to have the property ofbinding to hIL-6R using ATP as a switch. Regarding small moleculesbesides ATP, all clones were revealed to bind in the presence of ADP,and some clones were also found to bind in the presence of AMP and cAMP.In the presence of ADO (adenosine), hIL-6R binding was not observed.

(10-3) Assessment of Various Small Molecules for their Effect on theBinding to Human IL-6 Receptor by Octet

Octet (PRIMETECH) was used to assess various small molecules for theireffect on the antigen-antibody reaction between hIL-6R and nineATP/adenosine-dependent antibody clones (6RAD2C1-4_001, 6RAD2C1-4_005,6RAD2C1-4_011, 6RAD2C1-4_026, 6RAD2C1-4_030, 6RAD2C1-4_042,6RAD2C1-4_076, 6RDL3C1-4_085, and 6RDL3C5-4_011) obtained from alibrary. Assay was carried out at 30° C. using TBS, pH 7.4 as an assaybuffer. Biotinylated hIL-6R was immobilized onto a streptavidin sensorchip, and interacted with the antibodies to observe changes in theamount of binding. The antibodies were diluted with the assay buffer orthe assay buffer added with any one of ATP, ADP, AMP, cAMP, ADO, andATP-gamma-S. The final concentration of each small molecule was adjustedto 1 mM, and the final antibody concentration was adjusted to 10 μg/mL.

The assessment results on the hIL-6R binding of each clone in theabsence or presence of each small molecule at 1 mM obtained by thismeasurement demonstrated that all clones bound to hIL-6R in the presenceof 1 mM ATP. In the absence of ATP, hIL-6R binding was not observed.Thus, Octet also demonstrated that the clones had the property ofbinding to hIL-6R using ATP as a switch. Furthermore, binding wasobserved in the presence of ATP-gamma-S for all clones. The amount ofIL6R bound in the absence or presence of 1 mM ATP or ATP-gamma-Sobtained by the assessment is shown in FIG. 38. The findings describedabove demonstrate that hIL-6R-binding antibodies, for which ATP andderivatives thereof (ex-vivo molecules) function as a switch, could beisolated from a library.

Example 11 Obtaining Antibodies that Bind to Human IL-6 Receptor in thePresence of Kynurenine, by Panning with Kynurenine from a Library toIsolate Kynurenine-Switch Antibodies (Ver.A Kynurenine Library) (11-1)Obtaining Antibodies that have Binding Activity to Human IL-6 Receptorin the Presence of Kynurenine but not in the Absence of Kynurenine froman Antibody Library

The phage-display library for obtaining kynurenine-switch antibodies(referred to as the Ver.A kynurenine library), which was constructed asdescribed in Example 9 via panning with kynurenine, was screened forantibodies that have human IL-6 receptor (hIL-6R)-binding activity inthe presence of kynurenine. Specifically, phages presenting antibodiesthat have binding activity to hIL-6R captured on beads in the presenceof kynurenine, but are eluted from the beads in the absence ofkynurenine were collected. In this isolation method, biotin-labeledhIL-6R was used as an antigen.

E. coli retaining the constructed phage-display phagemid vector wasinfected with M13KO7ΔpIII (PROGEN Biotechnik; referred to ashyperphage), and cultured at 30° C. overnight. Phages were collectedfrom the supernatant. To precipitate the phage population, 2.5 MNaCl/10% PEG was added to the E. coli culture in which phages wereproduced. The phages were diluted with TBS to prepare a suspension ofantibody multivalent display phage library. Then, BSA was added at afinal concentration of 4% to the phage library suspension. Panning wasperformed using the antigen immobilized on magnetic beads. The magneticbeads used were NeutrAvidin coated beads (Sera-Mag SpeedBeadsNeutrAvidin-coated) or Streptavidin coated beads (Dynabeads MyOneStreptavidin T1).

The first panning was carried out to enrich phages that can bind to theantigen only in the presence of kynurenine. Specifically, together with250 pmol of the biotin-labeled antigen, kynurenine was added at a finalconcentration of 500 μM to 0.5 mL of the prepared phage librarysuspension, and thus the phage library was contacted with the antigenand kynurenine at room temperature for 15 minutes and then at 4° C. for45 minutes. BSA-blocked magnetic beads (Sera-Mag SpeedBeadsNeutrAvidin-coated) were added, and the antigen/phage complex wasallowed to bind to the magnetic beads at 4° C. for 15 minutes. The beadswere washed twice with 0.5 mL of ice-cold kynurenine/TBST and once withice-cold kynurenine/TBS. Immediately after the beads to which 0.25 mL ofTBS has been added were suspended at room temperature, the phagesuspension was collected from the beads that were separated using amagnetic stand. This process was repeated, and then the two phagesuspensions eluted separately were combined together. Five microliter of100 mg/mL trypsin was added to the collected phage suspension to cleavethe Fab. This improved the ability of trypsin-treated phages to infectE. coli. The trypsin-treated phages were added to 10 mL of E. colistrain ER2738 in the logarithmic growth phase (OD600=0.4-0.7). The E.coli was gently stirred and incubated at 37° C. for one hour to allowphages to infect the E. coli. The infected E. coli was seeded on a plateof 225 mm×225 mm. Then, E. coli in the seeded culture was infected withhyperphage, and cultured at 30° C. overnight. Phages were collected fromthe supernatant to prepare a suspension of antibody multivalent displayphage library.

In the second panning, together with kynurenine at a final concentrationof 500 μM, 250 pmol of the biotin-labeled antigen was added to 0.4 mL ofthe prepared phage library suspension, and thus the phage library wascontacted with the antigen and kynurenine at room temperature for 60minutes. BSA-blocked magnetic beads (Sera-Mag SpeedBeadsNeutrAvidin-coated) were added, and the antigen/phage complex wasallowed to bind to the magnetic beads at room temperature for 15minutes. The beads were washed twice with 0.5 mL of kynurenine/TBST andonce with kynurenine/TBS. Immediately after the beads to which 0.25 mLof TBS has been added were suspended at room temperature, the phagesuspension was collected from the beads that were separated using amagnetic stand. This process was repeated, and then the two phagesuspensions eluted separately were combined together. Five microliter of100 mg/mL trypsin was added to the collected phage suspension to cleavethe Fab. This improved the ability of trypsin-treated phages to infectE. coli. The trypsin-treated phages were added to 20 mL of E. colistrain ER2738 in the logarithmic growth phase (OD600=0.4-0.7). The E.coli was gently stirred and incubated at 37° C. for one hour to allowphages to infect the E. coli. The infected E. coli was seeded on a plateof 225 mm×225 mm. Then, E. coli in the seeded culture was infected withhyperphage, and cultured at 30° C. overnight. Phages were collected fromthe supernatant to prepare a suspension of antibody multivalent displayphage library.

In the third panning, together with kynurenine at a final concentrationof 500 nM, 100 pmol of the biotin-labeled antigen was added to 0.2 mL ofthe prepared phage library suspension, and thus the phage library wascontacted with the antigen and kynurenine at room temperature for 60minutes. BSA-blocked magnetic beads (Dynabeads MyOne Streptavidin T1)were added, and the antigen/phage complex was allowed to bind tomagnetic beads at 4° C. for 30 minutes. The beads were washed threetimes with 0.4 mL of kynurenine/TBST and twice with kynurenine/TBS.Immediately after the beads to which 0.25 mL of TBS has been added weresuspended at room temperature, the phage suspension was collected fromthe beads that were separated using a magnetic stand. This process wasrepeated, and then the two phage suspensions eluted separately werecombined together. Five microliter of 100 mg/mL trypsin was added to thecollected phage suspension to cleave the Fab. This improved the abilityof the trypsin-treated phages to infect E. coli. The trypsin-treatedphages were added to 20 mL of E. coli strain ER2738 in the logarithmicgrowth phase (OD600=0.4-0.7). The E. coli was gently stirred andincubated at 37° C. for one hour to allow phages to infect the E. coli.The infected E. coli was seeded on a plate of 225 mm×225 mm. Then, E.coli in the seeded culture was infected with hyperphage, and cultured at30° C. overnight. Phages were collected from the supernatant to preparea suspension of antibody multivalent display phage library.

The fourth panning was performed under the same conditions as the thirdpanning.

(11-2) Obtaining Antibodies that Bind to hIL-6R in the Presence ofKynurenine from an Antibody Library by a Negative Selection Method

The Ver.A kynurenine library constructed as described in Example 9 wasscreened for antibodies that have antigen-binding activity in thepresence of kynurenine. For screening, the antibody-phage-displaylibrary was first contacted with the biotin-labeled antigen-streptavidinin the absence of kynurenine to remove phages presenting antibodies thathave antigen-binding activity in the absence of kynurenine. Then, underthe condition where kynurenine is present, panning was performed in thesame manner to screen for antibodies having antigen-binding activityonly under the condition where kynurenine is present.

E. coli retaining the constructed phage-display phagemid vector wasinfected with M13KO7ΔpIII (PROGEN Biotechnik; referred to ashyperphage), and cultured at 30° C. overnight. Phages were collectedfrom the supernatant. To precipitate the population of phages, 2.5 MNaCl/10% PEG was added to the E. coli culture in which phages wereproduced. The phages were diluted with TBS to prepare a suspension ofantibody multivalent display phage library. Then, BSA was added at afinal concentration of 4% to the phage library suspension. Panning wasperformed using the antigen immobilized on magnetic beads. The magneticbeads used were NeutrAvidin coated beads (Sera-Mag SpeedBeadsNeutrAvidin-coated) or Streptavidin coated beads (Dynabeads MyOneStreptavidin T1).

In the first panning, phages that can bind to the antigen only in thepresence of kynurenine were enriched using a negative selection method.Specifically, 500 pmol of the biotinylated antigen was added toBSA-blocked Sera-Mag SpeedBeads NeutrAvidin-coated, and allowed to bindat room temperature for 15 minutes. After the beads were washed threetimes with TBS, they were combined with 0.5 mL of phage librarysuspension blocked with BSA and allowed to bind at room temperature forone hour. The beads were separated using a magnetic stand to collectphages that did not bind to the antigen or beads. Together with 250 pmolof the biotin-labeled antigen, kynurenine was added at a finalconcentration of 500 μM to the collected phages, and thus the phagelibrary was contacted with the antigen and kynurenine at roomtemperature for 15 minutes and then at 4° C. for 45 minutes. Then,BSA-blocked magnetic beads were added to the mixed solution of thelabeled antigen, kynurenine, and phage library; and the antigen/phagecomplex was allowed to bind to the magnetic beads at 4° C. for 15minutes. The beads were washed twice with 0.5 mL of ice-coldkynurenine/TBST and once with ice-cold kynurenine/TBS. Then, 0.5 mL of 1mg/mL trypsin solution was added to the mixture. The mixture was stirredfor 15 minutes at room temperature, and then phages were collected fromthe beads separated using a magnetic stand. The collected phages wereadded to 10 mL of E. coli strain ER2738 in the logarithmic growth phase(OD600=0.4-0.7). The E. coli was gently stirred and incubated at 37° C.for one hour to allow phages to infect the E. coli. The infected E. coliwas seeded on a plate of 225 mm×225 mm. Then, E. coli in the seededculture was infected with hyperphage, and cultured at 30° C. overnight.Phages were collected from the supernatant to prepare a suspension ofantibody multivalent display phage library.

The second and subsequent panning rounds were carried out to enrichphages that can bind to the antigen only in the presence of kynurenine.Specifically, panning was carried out up to the fourth round by the samemethod as used in the second and subsequent panning rounds described inExample (11-1).

(11-3) Obtaining Antibodies that Bind to hIL-6R in the Presence ofKynurenine from an Antibody Library by Panning with a Molecule Servingas a Switch

The Ver.A kynurenine library constructed as described in Example 9 wasscreened for antibodies that have antigen-binding activity under thecondition where kynurenine is present. For screening, first, panning wascarried out with biotinylated kynurenine (compounds 028, 029, and 036)to collect phages presenting antibodies that have kynurenine-bindingactivity. Then, in the presence of kynurenine, panning was performedwith biotin-labeled antigen to screen for antibodies that haveantigen-binding activity under the condition where kynurenine ispresent.

E. coli retaining the constructed phage-display phagemid vector wasinfected with M13KO7ΔpIII (PROGEN Biotechnik; referred to ashyperphage), and cultured at 30° C. overnight. Phages were collectedfrom the supernatant. To precipitate the population of phages, 2.5 MNaCl/10% PEG was added to the E. coli culture in which phages wereproduced. The phages were diluted with TBS to prepare a suspension ofantibody multivalent display phage library. Then, BSA was added at afinal concentration of 4% to the phage library suspension. Panning wasperformed using the antigen immobilized on magnetic beads. The magneticbeads used were NeutrAvidin coated beads (Sera-Mag SpeedBeadsNeutrAvidin-coated) or Streptavidin coated beads (Dynabeads MyOneStreptavidin T1).

In the first panning, phages that can bind to biotinylated kynurenine (amixture of compounds 028, 029, and 036) were enriched. Specifically,4000 pmol of the biotinylated kynurenine mixture containing equalamounts of compounds 028, 029, and 036 was added to BSA-blocked beads(Dynabeads MyOne Streptavidin T1) and allowed to bind at roomtemperature for 30 minutes. After the beads were washed three times withTBS, they were combined with 0.8 mL of phage library suspension blockedwith BSA, and allowed to contact with biotinylated kynurenine for 30minutes at room temperature and then for another 30 minutes at 4° C. Thebeads were washed three times with 1 mL of ice-cold TBST and twice withice-cold TBS. Then, 0.5 mL of 1 mg/mL trypsin solution was added to themixed solution. Immediately after suspending the mixture at roomtemperature for 15 minutes, the phage suspension was collected from thebeads that were separated using a magnetic stand. The collected phageswere added to 100 mL of E. coli strain ER2738 in the logarithmic growthphase (OD600=0.4-0.7). The E. coli was gently stirred and incubated at37° C. for one hour to allow phages to infect the E. coli. The infectedE. coli was seeded on five plates of 225 mm×225 mm. Then, E. coli in theseeded culture was infected with hyperphage, and cultured at 30° C.overnight. Phages were collected from the supernatant to prepare asuspension of antibody multivalent display phage library.

In the second and subsequent panning rounds, phages that can bind to theantigen only in the presence of kynurenine were enriched. Specifically,panning was carried out up to the fourth round by the same method asused in the second and subsequent panning rounds described in Example(11-1).

(11-4) Assessment of Binding Activity in the Presence of Kynurenine byPhage ELISA

Phages were cultured according to a conventional method (Methods Mol.Biol. (2002) 178, 133-145) from a single colony of E. coli obtained asdescribed in (11-1), (11-2), and (11-3). Culture supernatants containingantibody multivalent display phages were collected using hyperphages ashelper phages. The collected phage culture supernatants were subjectedto ultrafiltration using NucleoFast96 (MACHERY-NAGEL). 200 μL each ofthe culture supernatants were applied to each well of NucleoFast96. Theflowthrough fractions were removed via centrifugation at 6000 g for 45minutes. 200 μL of H₂O was added and the wells were washed bycentrifugation at 6000 g for 30 minutes. Then, 200 μL of TBS was added,and after allowing to stand for five minutes at room temperature, phagesuspensions contained in the supernatants were collected.

TBS or 500 μM kynurenine/TBS was added to the purified phages, and theywere subjected to ELISA by the procedure described below. A StreptaWell96 microtiter plate (Roche) was coated with 100 μL of TBS containingbiotin-labeled hIL-6R for one or more hours. After removingbiotin-labeled hIL-6R not bound to the plate by washing each well of theplate with TBST, the wells were blocked with 250 μL of 2% skim milk-TBSfor one or more hours. After removing 2% skim milk-TBS, the preparedpurified phages were added to each well of the plate. The plate wasallowed to stand at room temperature for one hour to allowantibody-presenting phages to bind to biotin-labeled hIL-6R in each wellin the presence/absence of kynurenine. After washing with TBST orkynurenine/TBST, HRP-conjugated anti-M13 antibody (Amersham PharmaciaBiotech) diluted with TBS or kynurenine/TBS was added to each well. Theplate was incubated for one hour. After washing with TBST orkynurenine/TBST, the chromogenic reaction of the solution in each welladded with TMB single solution (ZYMED) was terminated by adding sulfuricacid. Then, the color development was measured by absorbance at 450 nm.The results revealed several antibodies that bound to human IL-6receptor in the presence of kynurenine. The results of phage ELISA areshown in Table 35. As described in Example 6 above, antibodies that bindto human IL-6 receptor in the presence of kynurenine were also isolatedfrom the human naïve antibody library; however switch antibodies tohuman IL-6 receptor could be isolated with much greater efficiency. Theresults of phage ELISA carried out as described in Example 6-2 are shownin Table 36.

TABLE 35 Example Example Example Origin (11-1) (11-2) (11-3) Number ofpanning rounds 4 4 4 Number of clones subjected to 96 96 96 ELISA Numberof positive clones 96 94 94 (absorbance >0.2) Number of switch clones 9391 86 (kynurenine +/− absorbance ratio >2) Number of switch clonesequences 87 88 77

TABLE 36 Number of panning rounds 3 Number of clones subjected to ELISA960 Number of positive clones (absorbance >0.1) 939 Number of switchclones (kynurenine +/− absorbance ratio >1.5) 10 Number of switch clonesequences 2

(11-5) Sequence Analysis of Switch Antibodies Whose Antigen-BindingActivity Varies Depending on the Presence or Absence of Kynurenine

Using specific primers (SEQ ID NOs: 79 and 80), genes were amplifiedfrom clones judged to have antigen-binding activity under the conditionwhere kynurenine is present based on the phage ELISA results shown inExample (11-4). The nucleotide sequences of the genes were analyzed.From the analysis results, several clones judged to have bindingactivity to biotin-labeled hIL-6R in the presence of kynurenine wereisolated under all conditions described in Examples (11-1), (11-2), and(11-3). From the isolated clones, six clones were selected, whose aminoacid sequences and panning conditions for isolation (indicated as Originin the table) are shown in Table 37 below.

TABLE 37 Heavy chain Light chain Clone name Origin SEQ ID NO SEQ ID NO6RFHm12-4_040 Example (11-3) SEQ ID NO: 101 SEQ ID NO: 102 6RFHm12-4_078Example (11-3) SEQ ID NO: 103 SEQ ID NO: 104 6RFHm14-4_087 Example(11-2) SEQ ID NO: 105 SEQ ID NO: 106 6RFHm14-4_093 Example (11-2) SEQ IDNO: 107 SEQ ID NO: 108 6RFHm17-4_006 Example (11-1) SEQ ID NO: 109 SEQID NO: 110 6RFHm17-4_010 Example (11-1) SEQ ID NO: 111 SEQ ID NO: 112

(11-6) Expression and Purification of Antibodies that Bind to hIL-6R

The heavy chain and light chain variable region sequences of the 6clones described in Example (11-5) were inserted into a plasmid foranimal expression having an antibody heavy chain constant region (SEQ IDNO: 113) or light chain kappa constant region sequence (SEQ ID NO: 47).The antibodies were expressed and purified by the method described inReference Example 1 below.

(11-7) Assessment of the Obtained Antibodies for their hIL-6R-BindingActivity

The selected six types of antibodies and the 6RNMSC1-2_F02 antibody weresubjected to ELISA under the conditions indicated in Table 38.

TABLE 38 Antigen Kynurenine Tryptophan Condition 1 hIL6R 500 uM —Condition 2 hIL6R — — Condition 3 — 500 uM — Condition 4 hIL6R — 500 uM

First, a StreptaWell 96 microtiter plate (Roche) was coated with 100 μLof TBS containing biotin-labeled hIL-6R at room temperature for one ormore hours. After removing biotin-labeled hIL-6R that are not bound tothe plate by washing each well of the plate with TBST, the wells wereblocked with 250 μL of Blocking Buffer (2% skim milk/TBS) for one ormore hours. Blocking Buffer was removed from each well. 100 μL each ofthe purified IgGs prepared to 2.5 μg/mL using TBS under the conditionsdescribed in Table 38 were added to the wells, and the plate was allowedto stand at room temperature for one hour to allow each IgG to bind tobiotin-labeled hIL-6R in each well. TBST was prepared to make the finalconcentrations shown in Table 38, and each well was washed with theprepared TBST. HRP-conjugated anti-human IgG antibody (BIOSOURCE)diluted with TBS containing the same small molecules was added to eachwell. The plate was incubated for one hour. After washing with TBSTprepared to the final concentrations shown in Table 38, the chromogenicreaction of the solution in each well added with TMB single solution(ZYMED) was terminated by adding sulfuric acid. Then, the colordevelopment was measured by absorbance at 450 nm. The measurementresults are shown in FIG. 39.

The absorbance under condition 2 or 3 was markedly low as compared tothe absorbance under condition 1 in the selected six types ofantibodies, similarly to antibody 6RNMSC1-2_F02. This confirmed that theselected six types of antibodies in the form of IgG also have specificbinding activity against the biotin-labeled hIL-6R in the presence ofkynurenine. Meanwhile, no significant difference was observed for theabsorbance under condition 2 as compared to the absorbance undercondition 4. Thus, the selected six types of antibodies were alsoconfirmed to not have binding activity to the biotin-labeled hIL-6R inthe presence of tryptophan. The results described above shows thatmultiple hIL-6R-binding antibodies for which kynurenine serves as aswitch can be obtained from a library for isolating kynurenine-switchantibodies by using panning with kynurenine (Ver.A kynurenine library).Furthermore, as shown in Reference Example 4 below, antibodies that bindto a target antigen in the absence of ATP but do not bind to a targetantigen in the presence of ATP were obtained from the rationallydesigned library constructed using an ATP-binding antibody as a templateas described in Reference Example 2. This suggests that antibodies thatbind to a target antigen in the absence of kynurenine but do not bind toa target antigen in the presence of kynurenine can be similarly obtainedfrom a library for obtaining kynurenine-switch antibodies.

Example 12 Obtaining Antibodies that Bind to Human IgA-Fc (hIgA-Fc) inthe Presence of Kynurenine from a Library for ObtainingKynurenine-Switch Antibodies Using Panning with Kynurenine (Ver.AKynurenine Library) (12-1) Obtaining Antibodies that havehIgA-Fc-Binding Activity in the Presence of Kynurenine but not in theAbsence of Kynurenine from an Antibody Library

The Ver.A kynurenine library constructed as described in Example 9 wasscreened for antibodies that have human IgA-Fc (hIgA-Fc)-bindingactivity in the presence of kynurenine. Specifically, phages presentingantibodies that have binding activity to hIgA-Fc captured on beads inthe presence of kynurenine but are eluted from the beads in the absenceof kynurenine, were collected. In this method, biotin-labeled hIgA-Fc(SEQ ID NO: 146) prepared by the method described in Reference Example 5was used as an antigen.

E. coli retaining the constructed phage-display phagemid vector wasinfected with M13KO7ΔpIII (PROGEN Biotechnik; referred to ashyperphage), and cultured at 30° C. overnight. Phages were collectedfrom the supernatant. To precipitate the population of phages, 2.5 MNaCl/10% PEG was added to the E. coli culture in which phages wereproduced. The phages were diluted with TBS to prepare a suspension ofantibody multivalent display phage library. Then, BSA was added at afinal concentration of 4% to the phage library suspension. Panning wasperformed using the antigen immobilized on magnetic beads. The magneticbeads used were NeutrAvidin coated beads (Sera-Mag SpeedBeadsNeutrAvidin-coated) or Streptavidin coated beads (Dynabeads MyOneStreptavidin T1).

Panning was carried out to enrich phages that can bind to the antigenonly in the presence of kynurenine. Specifically, panning was carriedout up to the fourth round by a method similar to that indicated inExample (11-1), except that the antigen amount was changed to 500 pmolin the first and second rounds, and 200 pmol in the third and fourthrounds.

(12-2) Obtaining Antibodies that Bind to hIgA-Fc in the Presence ofKynurenine from an Antibody Library by a Negative Selection Method

The Ver.A kynurenine library constructed as described in Example 9 wasscreened for antibodies that have antigen-binding activity under thecondition where kynurenine is present. For screening, theantibody-phage-display library was first contacted with biotin-labeledantigen-streptavidin in the absence of kynurenine to remove phagespresenting antibodies that have antigen-binding activity in the absenceof kynurenine. Then, under the condition where kynurenine is present,panning was performed in the same manner to screen for antibodies havingantigen-binding activity only under the condition where kynurenine ispresent.

E. coli retaining the constructed phage-display phagemid vector wasinfected with M13KO7ΔpIII (PROGEN Biotechnik; referred to ashyperphage), and cultured at 30° C. overnight. Phages were collectedfrom the supernatant. To precipitate the population of phages, 2.5 MNaCl/10% PEG was added to the E. coli culture in which phages wereproduced. The phages were diluted with TBS to prepare a suspension ofantibody multivalent display phage library. Then, BSA was added at afinal concentration of 4% to the phage library suspension. Panning wasperformed using the antigen immobilized on magnetic beads. The magneticbeads used were NeutrAvidin coated beads (Sera-Mag SpeedBeadsNeutrAvidin-coated) or Streptavidin coated beads (Dynabeads MyOneStreptavidin T1).

In the first panning, phages that can bind to the antigen only in thepresence of kynurenine were enriched using a negative selection method.Specifically, 1000 pmol of the biotinylated antigen was added toBSA-blocked Sera-Mag SpeedBeads NeutrAvidin-coated and allowed to bindthereto at room temperature for 15 minutes. After the beads were washedthree times with TBS, they were combined with 0.5 ml of phage librarysuspension blocked with BSA and allowed to bind at room temperature forone hour. The beads were separated using a magnetic stand to collectphages that did not bind to the antigen or beads. Together with 500 pmolof the biotin-labeled antigen, kynurenine was added at a finalconcentration of 500 μM to the collected phages, and thus the phagelibrary was contacted with the antigen and kynurenine at roomtemperature for 15 minutes and then at 4° C. for 45 minutes. Then,BSA-blocked magnetic beads were added to the mixed solution of thelabeled antigen, kynurenine, and phage library, and the antigen/phagecomplex was allowed to bind to the magnetic beads at 4° C. for 15minutes. The beads were washed twice with 0.5 ml of ice-coldkynurenine/TBST and once with ice-cold kynurenine/TBS. Then, 0.5 ml of 1mg/ml trypsin solution was added to the mixed solution. After themixture was stirred for 15 minutes at room temperature, phages werecollected from the beads separated using a magnetic stand. The collectedphages were added to 10 ml of E. coli strain ER2738 in the logarithmicgrowth phase (OD600=0.4-0.7). The E. coli was gently stirred andincubated at 37° C. for one hour to allow phages to infect the E. coli.The infected E. coli was seeded on a plate of 225 mm×225 mm. Then, E.coli in the seeded culture was infected with hyperphage, and cultured at30° C. overnight. Phages were collected from the supernatant to preparea suspension of antibody multivalent display phage library.

The second and subsequent panning rounds were carried out to enrichphages that can bind to the antigen only in the presence of kynurenine.Specifically, panning was carried out up to the fourth round by a methodsimilar to the second and subsequent panning rounds described in Example(11-1), except that the antigen amount was changed to 500 pmol in thesecond round and 200 pmol in the third and fourth rounds.

(12-3) Obtaining Antibodies that Bind to hIgA-Fc in the Presence ofKynurenine, from an Antibody Library by Panning with a Molecule Servingas a Switch

The Ver.A kynurenine library constructed as described in Example 9 wasscreened for antibodies that have antigen-binding activity under thecondition where kynurenine is present. For screening, panning was firstcarried out with biotinylated kynurenine (compounds 028, 029, and 036)to collect phages presenting antibodies that have kynurenine-bindingactivity. Then, panning was performed with biotin-labeled antigen in thepresence of kynurenine to screen for antibodies that haveantigen-binding activity under the condition where kynurenine ispresent.

E. coli retaining the constructed phage-display phagemid vector wasinfected with M13KO7ΔpIII (PROGEN Biotechnik; referred to ashyperphage), and cultured at 30° C. overnight. Phages were collectedfrom the supernatant. To precipitate a population of phages, 2.5 MNaCl/10% PEG was added to the E. coli culture in which phages wereproduced. The phages were diluted with TBS to prepare a suspension ofantibody multivalent display phage library. Then, BSA was added at afinal concentration of 4% to the phage library suspension. Panning wasperformed using the antigen immobilized on magnetic beads. The magneticbeads used were NeutrAvidin coated beads (Sera-Mag SpeedBeadsNeutrAvidin-coated) or Streptavidin coated beads (Dynabeads MyOneStreptavidin T1).

The first panning was carried out to enrich phages that can bind tobiotinylated kynurenine (a mixture of compounds 028, 029, and 036).Specifically, 4000 pmol of a biotinylated kynurenine mixture containingequal amounts of compounds 028, 029, and 036 was added to BSA-blockedbeads (Dynabeads MyOne Streptavidin T1) and allowed to bind at roomtemperature for 30 minutes. After washing the beads three times withTBS, they were combined with 0.8 ml of phage library suspension blockedwith BSA, and allowed to contact with biotinylated kynurenine for 30minutes at room temperature and then for 30 minutes at 4° C. The beadswere washed three times with 1 ml of ice-cold TBST and twice withice-cold TBS. Then, 0.5 ml of 1 mg/ml trypsin solution was added to themixed solution. Immediately after the mixture was suspended at roomtemperature for 15 minutes, a phage suspension was collected from thebeads that were separated using a magnetic stand. The collected phageswere added to 100 ml of E. coli strain ER2738 in the logarithmic growthphase (OD600=0.4-0.7). The E. coli was gently stirred and incubated at37° C. for one hour to allow phages to infect the E. coli. The infectedE. coli was seeded on five plates of 225 mm×225 mm. Then, E. coli in theseeded culture was infected with hyperphage, and cultured at 30° C.overnight. Phages were collected from the supernatant to prepare asuspension of antibody multivalent display phage library.

The second and subsequent panning rounds were carried out to enrichphages that can bind to the antigen only in the presence of kynurenine.Specifically, panning was carried out up to the fourth round by a methodsimilar to the second and subsequent panning rounds described in Example(11-1), except that the antigen amount was changed to 500 pmol in thesecond round and 200 pmol in the third and fourth rounds.

(12-4) Assessment of Binding Activity in the Presence of Kynurenine byPhage ELISA

Phages were cultured according to a conventional method (Methods Mol.Biol. (2002) 78, 133-145) from a single colony of E. coli obtained asdescribed in (12-1), (12-2), and (12-3). A culture supernatantcontaining antibody multivalent display phages was collected usinghyperphages as helper phages. Phages purified by the method described inExample (11-4) were subjected to ELISA by the procedure described below.A StreptaWell 96 microtiter plate (Roche) was coated with 100 μl of TBScontaining biotin-labeled hIgA-Fc for one or more hours. Each well ofthe plate was washed with TBST to remove biotin-labeled hIgA-Fc that isnot bound to the plate, and the wells were blocked with 250 μl of 2%skim milk-TBS for one or more hours. After removing 2% skim milk-TBS,the prepared purified phages were added to each well. The plate wasallowed to stand at room temperature for one hour to allowantibody-presenting phages to bind to biotin-labeled hIgA-Fc in eachwell in the presence/absence of kynurenine. Each well was washed withTBST or kynurenine/TBST, and HRP-conjugated anti-M13 antibody (AmershamPharmacia Biotech) diluted with TBS or kynurenine/TBS was added thereto.The plate was incubated for one hour. After washing with TBST orkynurenine/TBST, the chromogenic reaction of the solution in each welladded with TMB single solution (ZYMED) was terminated by adding sulfuricacid. Then, color development was measured by absorbance at 450 nm. As aresult, several antibodies were confirmed to bind to human IgA-Fc in thepresence of kynurenine. The results of phage ELISA are shown in Table39.

TABLE 39 Example Example Example Origin (12-1) (12-2) (12-3) Number ofpanning rounds 4 4 4 Number of clones subjected to 96 96 96 ELISA Numberof positive clones 89 90 91 (absorbance >0.2) Number of switch clones 8790 86 (kynurenine +/− absorbance ratio >2) Number of switch clonesequences 67 69 75

(12-5) Sequence Analysis of Switch Antibodies Whose Antigen-BindingActivity Varies Depending on the Presence or Absence of Kynurenine

Using specific primers (SEQ ID NOs: 79 and 80), genes were amplifiedfrom clones judged to have antigen-binding activity under the conditionwhere kynurenine is present based on the results of phage ELISA shown inExample (12-4). The nucleotide sequences of the genes were analyzed, andas a result, several clones judged to have binding activity to thebiotin-labeled hIgA-Fc in the presence of kynurenine were obtained undereach condition described in Examples (12-1), (12-2), and (12-3). Fromthe obtained clones, six clones were selected, whose amino acidsequences and panning conditions for obtainment (indicated as Origin inthe table) are shown in Table 40 below.

TABLE 40 Clone name Origin Heavy chain SEQ ID NO Light chain SEQ ID NOhIAFHm12-4_018 Example (12-3) SEQ ID NO: 114 SEQ ID NO: 115hIAFHm12-4_061 Example (12-3) SEQ ID NO: 116 SEQ ID NO: 117hIAFHm14-4_001 Example (12-2) SEQ ID NO: 118 SEQ ID NO: 119hIAFHm14-4_041 Example (12-2) SEQ ID NO: 120 SEQ ID NO: 121hIAFHm17-4_026 Example (12-1) SEQ ID NO: 122 SEQ ID NO: 123hIAFHm17-4_072 Example (12-1) SEQ ID NO: 124 SEQ ID NO: 125

(12-6) Expression and Purification of Antibodies that Bind to hIgA-Fc

Each of the heavy chain and light chain variable region sequences of thesix clones described in Example (12-5) were inserted into a plasmid foranimal expression having an antibody heavy chain constant region (SEQ IDNO: 113) or light chain kappa constant region sequence (SEQ ID NO: 47).Antibodies were expressed and purified by the method described inReference Example 1 below.

(12-7) Assessment of Obtained Antibodies for their hIgA-Fc-BindingActivity

The obtained six types of antibodies were subjected to ELISA under theconditions described in Table 41.

TABLE 41 Antigen Kynurenine Tryptophan Condition 1 hIgA-Fc 500 uM —Condition 2 hIgA-Fc — — Condition 3 — 500 uM — Condition 4 hIgA-Fc — 500uM

First, a StreptaWell 96 microtiter plate (Roche) was coated with 100 μlof TBS containing biotin-labeled hIgA-Fc at room temperature for one ormore hours. Each well of the plate was washed with TBST to removebiotin-labeled hIgA-Fc not bound to the plate and the wells were blockedwith 250 μl of Blocking Buffer (2% skim milk/TBS) for one or more hours.Blocking Buffer was removed from each well. 100 μl each of the purifiedIgGs prepared to 2.5 μg/ml with TBS under the conditions shown in Table41 were added to the plate. The plate was left to stand at roomtemperature for one hour to allow each IgG to bind to the biotin-labeledhIgA-Fc in each well. Each well was washed with TBST prepared at a finalconcentration shown in Table 41. HRP-conjugated anti-human IgG antibody(BIOSOURCE) diluted with TBS containing the same small molecules wasadded to the wells. The plate was incubated for one hour. After washingwith TBST prepared to the final concentrations shown in Table 41, thechromogenic reaction of the solution in each well added with TMB singlesolution (ZYMED) was terminated by adding sulfuric acid. Then, colordevelopment was measured by absorbance at 450 nm. The measurementresults are shown in FIG. 40.

Regarding the selected six types of antibodies, the absorbance undercondition 2 or 3 was markedly low as compared to the absorbance undercondition 1. This finding confirms that the selected six types ofantibodies, in the form of IgG, also have specific binding activity tobiotin-labeled hIgA-Fc in the presence of kynurenine. Meanwhile, theabsorbance under condition 2 was not markedly different as compared tothe absorbance under condition 4. Thus, the selected six types ofantibodies were also confirmed to not have binding activity tobiotin-labeled hIgA-Fc in the presence of tryptophan. The resultsdescribed above shows that a number of hIgA-Fc-binding antibodies forwhich kynurenine functions as a switch can be obtained from a libraryfor obtaining kynurenine-switch antibodies using panning with kynurenine(Ver. A kynurenine library).

Example 13 Obtaining Antibodies that Bind to Human IL-6 (hIL-6) in thePresence of Kynurenine from a Library for Obtaining Kynurenine-SwitchAntibodies Using Panning with Kynurenine (Ver. A Kynurenine Library)(13-1) Obtaining Antibodies that have Binding Activity to hIL-6 in thePresence of Kynurenine but do not Bind in the Absence of Kynurenine froman Antibody Library

The Ver.A kynurenine library constructed as described in Example 9 wasscreened for antibodies having binding activity to human IL-6 (hIL-6) inthe presence of kynurenine. Namely, phages presenting antibodies thathave binding activity to hIL-6 captured on beads in the presence ofkynurenine, but that are eluted from the beads in the absence ofkynurenine, were collected. In this isolation method, biotin-labeledhIL-6 was used as an antigen.

E. coli retaining the constructed phage-display phagemid vector wasinfected with M13KO7ΔpIII (PROGEN Biotechnik; referred to ashyperphage), and cultured at 30° C. overnight. Phages were collectedfrom the supernatant. To precipitate a population of phages, 2.5 MNaCl/10% PEG was added to the E. coli culture in which phages wereproduced. The phages were diluted with TBS to prepare a suspension ofantibody multivalent display phage library. Then, BSA was added at afinal concentration of 4% to the phage library suspension. Panning wasperformed using the antigen immobilized on magnetic beads. The magneticbeads used were NeutrAvidin coated beads (Sera-Mag SpeedBeadsNeutrAvidin-coated) or Streptavidin coated beads (Dynabeads MyOneStreptavidin T1).

Panning was carried out to enrich phages that can bind to the antigenonly in the presence of kynurenine. Panning was carried out up to thefourth round by a method similar to that described in Example (11-1).

(13-2) Obtaining Antibodies that Bind to hIL-6 in the Presence ofKynurenine Using a Negative Selection Method from an Antibody Library

The Ver.A kynurenine library constructed as described in Example 9 wasscreened for antibodies that have antigen-binding activity under thecondition where kynurenine is present. For screening purpose, theantibody-phage-display library was first contacted with biotin-labeledantigen-streptavidin in the absence of kynurenine to remove phagespresenting antibodies that have antigen-binding activity even in theabsence of kynurenine. Then, panning was similarly performed under thecondition where kynurenine is present to screen for antibodies havingantigen-binding activity under the condition where kynurenine ispresent.

E. coli retaining the constructed phage-display phagemid vector wasinfected with M13KO7ΔpIII (PROGEN Biotechnik; referred to ashyperphage), and cultured at 30° C. overnight. Phages were collectedfrom the supernatant. To precipitate a population of phages, 2.5 MNaCl/10% PEG was added to the E. coli culture in which phages wereproduced. The phages were diluted with TBS to prepare a suspension ofantibody multivalent display phage library. Then, BSA was added at afinal concentration of 4% to the phage library suspension. Panning wasperformed using the antigen immobilized on magnetic beads. The magneticbeads used were NeutrAvidin coated beads (Sera-Mag SpeedBeadsNeutrAvidin-coated) or Streptavidin coated beads (Dynabeads MyOneStreptavidin T1).

In the first round of panning, phages that can bind to the antigen onlyin the presence of kynurenine were enriched using a negative selectionmethod. Specifically, 500 pmol of biotinylated antigen was added toBSA-blocked Sera-Mag SpeedBeads NeutrAvidin-coated and allowed to bindat room temperature for 15 minutes. After washing the beads three timeswith TBS, they were combined with 0.5 ml of phage library suspensionblocked with BSA and allowed to bind at room temperature for one hour.The beads were separated using a magnetic stand to collect phages thatdid not bind to the antigen or beads. Together with 250 pmol ofbiotin-labeled antigen, kynurenine was added at a final concentration of500 μM to the collected phages, and the phage library was contacted withthe antigen and kynurenine at room temperature for 15 minutes and thenat 4° C. for 45 minutes. Then, BSA-blocked magnetic beads were added tothe mixed solution of the labeled antigen, kynurenine, and phagelibrary, and the antigen/phage complex was allowed to bind to themagnetic beads at 4° C. for 15 minutes. The beads were washed twice with0.5 ml of ice-cold kynurenine/TBST and once with ice-coldkynurenine/TBS. Then, 0.5 ml of 1 mg/ml trypsin solution was added tothe mixed solution. The mixed solution was stirred for 15 minutes atroom temperature, and then phages were collected from the beadsseparated using a magnetic stand. The collected phages were added to 10ml of E. coli strain ER2738 in the logarithmic growth phase(OD600=0.4-0.7). The E. coli was gently stirred and incubated at 37° C.for one hour to allow phages to infect the E. coli. The infected E. coliwas seeded on a plate of 225 mm×225 mm. Then, E. coli in the seededculture was infected with hyperphage, and cultured at 30° C. overnight.Phages were collected from the supernatant to prepare a suspension ofantibody multivalent display phage library.

In the second and subsequent panning rounds, phages that can bind to theantigen only in the presence of kynurenine were enriched. Specifically,panning was carried out up to the fourth round by a method similar tothe second and subsequent panning rounds described in Example (11-1).

(13-3) Obtaining Antibodies that Bind to hIL-6 in the Presence ofKynurenine by Panning with a Molecule Serving as a Switch, from anAntibody Library

The Ver.A kynurenine library constructed as described in Example 9 wasscreened for antibodies that have antigen-binding activity under thecondition where kynurenine is present. For screening, panning was firstcarried out with biotinylated kynurenine (compounds 028, 029, and 036)to collect phages presenting antibodies that have kynurenine-bindingactivity. Then, panning was performed with biotin-labeled antigen in thepresence of kynurenine to screen for antibodies having antigen-bindingactivity under the condition where kynurenine is present.

E. coli retaining the constructed phage-display phagemid vector wasinfected with M13KO7ΔpIII (PROGEN Biotechnik; referred to ashyperphage), and cultured at 30° C. overnight. Phages were collectedfrom the supernatant. To precipitate a population of phages, 2.5 MNaCl/10% PEG was added to the E. coli culture in which phages wereproduced. The phages were diluted with TBS to prepare a suspension ofantibody multivalent display phage library. Then, BSA was added at afinal concentration of 4% to the phage library suspension. Panning wasperformed using the antigen immobilized on magnetic beads. The magneticbeads used were NeutrAvidin coated beads (Sera-Mag SpeedBeadsNeutrAvidin-coated) or Streptavidin coated beads (Dynabeads MyOneStreptavidin T1).

In the first round of panning, phages that can bind to biotinylatedkynurenine (a mixture of compounds 028, 029, and 036) were enriched.Specifically, 4000 pmol of a biotinylated kynurenine mixture containingequal amounts of compounds 028, 029, and 036 was added to BSA-blockedbeads (Dynabeads MyOne Streptavidin T1) and allowed to bind at roomtemperature for 30 minutes. The beads were washed three times with TBSand combined with 0.8 ml of phage library suspension blocked with BSA,and allowed to contact with biotinylated kynurenine for 30 minutes atroom temperature and then 30 minutes at 4° C. The beads were washedthree times with 1 ml of ice-cold TBST and twice with ice-cold TBS.Then, 0.5 ml of 1 mg/ml trypsin solution was added to the mixedsolution. Immediately after the mixture was suspended at roomtemperature for 15 minutes, a phage suspension was collected from thebeads that were separated using a magnetic stand. The collected phageswere added to 100 ml of E. coli strain ER2738 in the logarithmic growthphase (OD600=0.4-0.7). The E. coli was gently stirred and incubated at37° C. for one hour to allow phages to infect the E. coli. The infectedE. coli was seeded on five plates of 225 mm×225 mm. Then, E. coli in theseeded culture was infected with hyperphage, and cultured at 30° C.overnight. Phages were collected from the supernatant to prepare asuspension of antibody multivalent display phage library.

In the second and subsequent panning rounds, phages that can bind to theantigen only in the presence of kynurenine were enriched. Specifically,panning was carried out up to the fourth round by a method similar tothe second and subsequent panning rounds described in Example (11-1).

(13-4) Assessment of Binding Activity in the Presence of Kynurenine byPhage ELISA

Phages were cultured according to a conventional method (Methods Mol.Biol. (2002) 78, 133-145) from a single colony of E. coli obtained asdescribed in (13-1), (13-2), and (13-3). A culture supernatantcontaining antibody multivalent display phages was collected usinghyperphages as helper phages. Phages purified by the method described inReference Example (11-4) were subjected to ELISA by the proceduredescribed below. A 384-well Microplates Streptavidin-coated (GreinerBio-One) was coated with 10 μl of TBS containing biotin-labeled hIL-6for one or more hours. Each well of the plate was washed with TBST toremove biotin-labeled hIL-6 not bound to the plate and the wells wereblocked with 80 μl of 2% skim milk-TBS for one or more hours. Afterremoving 2% skim milk-TBS, the prepared purified phages were added toeach well of the plate. The plate was left to stand at room temperaturefor one hour to allow antibody-presenting phages to bind tobiotin-labeled hIL-6 in each well in the presence/absence of kynurenine.Each well was washed with TBST or kynurenine/TBST. HRP-conjugatedanti-M13 antibody (Amersham Pharmacia Biotech) diluted with TBS orkynurenine/TBS was added to the wells. The plate was incubated for onehour. After washing with TBST or kynurenine/TBST, the chromogenicreaction of the solution in each well added with TMB single solution(ZYMED) was terminated by adding sulfuric acid. Then, the colordevelopment was measured by absorbance at 450 nm. As a result, severalantibodies were confirmed to bind to hIL-6 in the presence ofkynurenine. The results of phage ELISA are shown in Table 42.

TABLE 42 Example Example Example Origin (13-1) (13-2) (13-3) Number ofpanning rounds 4 4 4 Number of clones subjected to 96 96 95 ELISA Numberof positive clones 65 59 54 (absorbance >0.2) Number of switch clones 6158 48 (kynurenine +/− absorbance ratio >2) Number of switch clonesequences 42 37 44

(13-5) Sequence Analysis of Switch Antibodies Whose Antigen-BindingActivity Varies Depending on the Presence or Absence of Kynurenine

Using specific primers (SEQ ID NOs: 79 and 80), genes were amplifiedfrom clones judged to have antigen-binding activity under the conditionwhere kynurenine is present based on the phage ELISA results shown inExample (13-4). The nucleotide sequences of the genes were analyzed.From the analysis results, several clones judged to have bindingactivity to biotin-labeled hIL-6 in the presence of kynurenine wereobtained under each condition described in Examples (13-1), (13-2), and(13-3). From the obtained clones, six clones were selected and theiramino acid sequences and panning conditions for isolation (indicated asOrigin in the table) are shown in Table 43 below.

TABLE 43 Heavy chain Light chain Clone name Origin SEQ ID NO SEQ ID NO16FHm12-4_068 Example (13-3) SEQ ID NO: 126 SEQ ID NO: 127 16FHm12-4_094Example (13-3) SEQ ID NO: 128 SEQ ID NO: 129 16FHm14-4_007 Example(13-2) SEQ ID NO: 130 SEQ ID NO: 131 16FHm14-4_030 Example (13-2) SEQ IDNO: 132 SEQ ID NO: 133 16FHm17-4_016 Example (13-1) SEQ ID NO: 134 SEQID NO: 135 16FHm17-4_036 Example (13-1) SEQ ID NO: 136 SEQ ID NO: 137

(13-6) Expression and Purification of Antibodies that Bind to hIL-6

The heavy chain and light chain variable region sequences of the sixclones described in Example (13-5) were each inserted into a plasmid foranimal expression having an antibody heavy chain constant region (SEQ IDNO: 113) or a light chain kappa constant region sequence (SEQ ID NO:47). Expression and purification of the antibodies were performed by themethod described in Reference Example 1 below.

(13-7) Assessment of the Obtained Antibodies for their hIL-6-BindingActivity

The obtained six types of antibodies were subjected to ELISA under theconditions described in Table 44.

TABLE 44 Antigen Kynurenine Tryptophan Condition 1 hIL-6 500 uM —Condition 2 hIL-6 — — Condition 3 — 500 uM — Condition 4 hIL-6 — 500 uM

First, a StreptaWell 96 microtiter plate (Roche) was coated with 100 μlof TBS containing biotin-labeled hIL-6 at room temperature for one ormore hours. Each well of the plate was washed with TBST to removebiotin-labeled hIL-6 not bound to the plate and the wells were blockedwith 250 μl of Blocking Buffer (2% skim milk/TBS) for one or more hours.Blocking Buffer was removed from each well. 100 μl each of the purifiedIgGs prepared to 2.5 μg/ml with TBS under the conditions shown in Table44 were added to the well. The plate was left to stand at roomtemperature for one hour to allow each IgG to bind to biotin-labeledhIL-6 in each well. Each well was washed with TBST prepared at a finalconcentration shown in Table 44. HRP-conjugated anti-human IgG antibody(BIOSOURCE) diluted with TBS containing the same small molecules wasadded to the wells. The plate was incubated for one hour. After washingwith TBST prepared at a final concentration shown in Table 44, thechromogenic reaction of the solution in each well added with TMB singlesolution (ZYMED) was terminated by adding sulfuric acid. Then, the colordevelopment was measured by absorbance at 450 nm. The measurementresults are shown in FIG. 41.

Regarding the selected six types of antibodies, the absorbance undercondition 2 or 3 was markedly low as compared to the absorbance undercondition 1. This finding confirms that the selected six types ofantibodies, in the form of IgG, also have specific binding activity tobiotin-labeled hIL-6 in the presence of kynurenine. Meanwhile, theabsorbance under condition 2 was not markedly different as compared tothe absorbance under condition 4. Thus, the selected six types ofantibodies were also confirmed to not have binding activity tobiotin-labeled hIL-6 in the presence of tryptophan. The resultsdescribed above shows that a number of hIL-6-binding antibodies forwhich kynurenine functions as a switch can be obtained from a libraryfor obtaining kynurenine-switch antibodies, using panning withkynurenine (Ver.A kynurenine library).

Reference Example 1 Obtaining Antibodies that Bind to Adenosine and/orATP, from a Human Antibody Library by Phage Display Techniques (1-1)Preparation of Naïve Human Antibody Phage-Display Library

A human antibody phage-display library consisting of multiple phagesthat present the Fab domains of human antibody sequences that aredifferent from one another was constructed using, as a template, polyARNA prepared from human PBMC, commercially available human polyA RNA, orsuch according to a method known to those skilled in the art.

(1-2) Acquisition of Antibodies that Bind to Adenosine and/or ATP fromLibrary by Bead Panning

The phage-display library of naïve human antibodies constructed asdescribed in Reference Example (1-1) was screened for antibodies thatexhibit antigen-binding activity, specifically, by collecting phagesthat display antibodies with binding activity to antigens captured bybeads. ATP-PEG-Biotin, 2′-Adenosine-PEG-Biotin, and5′-Adenosine-PEG-Biotin were used as antigens. Regarding ATP-PEG-Biotin,Cat. No. NU-926-BIO was purchased from Jena Bioscience GmbH and used.Regarding 2′-Adenosine-PEG-Biotin and 5′-Adenosine-PEG-Biotin, thoseconstructed in Example (2-2-11) were used.

Phages produced in E. coli containing the phagemid vector constructedfor phage display were purified by a conventional method, and thendialyzed against TBS to prepare a phage library suspension. Then, BSAwas added at a final concentration of 4% to the suspension. Panning wasperformed using antigen-immobilized magnetic beads. The magnetic beadsused were NeutrAvidin coated beads (Sera-Mag SpeedBeadsNeutrAvidin-coated) and Streptavidin coated beads (Dynabeads M-280Streptavidin).

Next, 250 pmol of biotinylated ATP, 2′-adenosine-PEG-Biotin, and5′-adenosine-PEG-Biotin were added to the prepared phage librarysuspension. Thus, the phage library suspension was contacted withadenosine and ATP for 60 minutes at room temperature. Then, BSA-blockedmagnetic beads were added to the phage library suspension, and thecomplex of phage with adenosine and/or ATP was allowed to bind to themagnetic beads at room temperature for 15 minutes. The beads were washedonce with TBS. Then, the beads were combined with 0.5 ml of 1 mg/mltrypsin solution. Immediately after the beads were suspended at roomtemperature for 15 minutes, a phage suspension was collected from thebeads that were separated using a magnetic stand. The collected phagesuspension was added to 10 ml of E. coli cells of strain ER2738 at thelogarithmic growth phase (OD600=0.4 to 0.7). The E. coli was incubatedat 37° C. for one hour under gentle stirring to be infected by phage.The infected E. coli was seeded in a 225 mm×225 mm plate. Then, phageswere collected from the culture medium of the seeded E. coli to preparea liquid stock of phage library.

A second round of panning was performed to also enrich phages that arecapable of binding to adenosine and/or ATP. The prepared phage librarysuspension was contacted with adenosine and ATP for 60 minutes at roomtemperature by adding 50 pmol each of biotinylated ATP,2′-Adenosine-PEG-Biotin, and 5′-Adenosine-PEG-Biotin. Then, theBSA-blocked magnetic beads were added to the phage library suspension,and the complex of phage with adenosine and/or ATP was allowed to bindto the magnetic beads at room temperature for 15 minutes. The beads werewashed three times with TBST and twice with TBS. Then, the beads werecombined with 0.5 ml of 1 mg/ml trypsin solution. Immediately after thebeads were suspended at room temperature for 15 minutes, a phagesuspension was collected from the beads separated using a magneticstand. The collected phage suspension was added to 10 ml of E. colicells of strain ER2738 at the logarithmic growth phase (OD600=0.4 to0.7). The E. coli was incubated at 37° C. for one hour with gentlestirring to be infected by phage. The infected E. coli was seeded in a225 mm×225 mm plate. Then, phages were collected from the culture mediumof the seeded E. coli to prepare a liquid stock of phage library.

By the same procedure, panning was performed three times to obtainantibodies that are capable of binding to adenosine and/or ATP. In thefourth round of panning, TBST wash and TBS wash were each performed fivetimes.

(1-3) Assessment of Adenosine- and ATP-Binding Activity by Phage ELISA

From single colonies of E. coli obtained by panning as described in theExample above, culture supernatants containing phages were collectedaccording to a conventional method (Method Mol. Biol. (2002) 178,133-145). The collected culture supernatants were treated byultrafiltration using NucleoFast 96 (MACHERY-NAGEL). 100 μl of theculture supernatants were added to each well of NucleoFast 96 andcentrifuged at 4500 g for 45 minutes to remove flow through. Afteraddition of 100 μl of H₂O, the NucleoFast 96 was washed bycentrifugation at 4500 g for 30 minutes. After addition of 100 μl ofTBS, the NucleoFast 96 was allowed to stand for five minutes at roomtemperature. Then, phage suspensions were collected from thesupernatants.

Purified phages, to which TBS was added, were subjected to ELISA by thefollowing procedure. A StreptaWell 96 microtiter plate (Roche) wascoated at room temperature for one hour with 100 μl of TBS containingbiotin-labeled antigens (a mixture of equal amounts of2′-adenosine-PEG-biotin, 5′-adenosine-PEG-biotin, and ATP-PEG-biotin).After antigens were removed from each well of the plate by washing withTBST (TBS containing 0.1% Tween20), the wells were blocked with 250 μlof 2% skim milk/TBS for one hour or more. 2% skim milk/TBS was removed,and then the prepared, purified phages were added to each well. Theplate was allowed to stand at room temperature for one hour to allow theantibody-displaying phage to bind antigens in each well. After washingwith TBST, a HRP-conjugated anti-M13 antibody (Amersham PharmaciaBiotech) diluted with TBS was added to each well. The plate wasincubated for one hour. Following TBST wash, the TMB single solution(ZYMED) was added to each well. The chromogenic reaction in the solutionof each well was terminated by adding sulfuric acid. Then, the developedcolor was assessed by measuring absorbance at 450 nm.

From the 192 clones subjected to phage ELISA, 106 clones that have theability to bind to any one or two, or all three of2′-Adenosine-PEG-biotin, 5′-Adenosine-PEG-biotin, and ATP-PEG-biotinwere obtained.

Next, for the purpose of confirming to which antigen of2′-adenosine-PEG-biotin, 5′-adenosine-PEG-biotin, and ATP-PEG-biotinthese clones have binding ability, the purified phages diluted with TBSwere subjected to ELISA by the following procedure. A StreptaWell 96microtiter plate (Roche) was coated at room temperature for one hourwith 100 μl of TBS containing a biotin-labeled antigen(2′-adenosine-PEG-biotin, 5′-adenosine-PEG-biotin, or ATP-PEG-biotin).After the antigens were removed by washing each well of the plate withTBST, the wells were blocked with 250 μl of 2% skim milk/TBS for onehour or more. 2% skim milk/TBS was removed, and then the prepared,purified phages were added to each well. The plate was allowed to standat room temperature for one hour to allow binding of antibody-displayingphages to antigens in each well. After TBST wash, a HRP-conjugatedanti-M13 antibody (Amersham Pharmacia Biotech) diluted with TBS wasadded to each well. The plate was incubated for one hour. Following TBSTwash, the TMB single solution (ZYMED) was added to each well. Thechromogenic reaction in the solution of each well was terminated byadding sulfuric acid. Then, the developed color was assessed bymeasuring absorbance at 450 nm. The result of phage ELISA is shown inTable 45 below.

TABLE 45 Antigen binding ability (S/N Enrichment indicator ratio >1.5)Number of panning rounds 4 Number of clones subjected to ELISA 192Number Combination of 2′-Adenosine-PEG-biotin, 5′- 106 ofAdenosine-PEG-biotin, ATP-PEG-biotin ELISA- 2′-Adenosine-PEG-biotin 0positive 5′-Adenosine-PEG-biotin 6 clones ATP-PEG-biotin 76 Bind to twoor more of 2′-Adenosine-PEG-biotin, 1 5′-Adenosine-PEG-biotin, andATP-PEG-biotin

Among the clones subjected to phage ELISA, a clone was demonstrated tobind to two or more types of antigens. The nucleotide sequence of thegene was analyzed using the antibody fragment as a template. This clonehad the ability to bind to both 5′-Adenosine-PEG-biotin andATP-PEG-biotin, and was named ATNLSA1-4_D12. The heavy-chain variableregion sequence of antibody ATNLSA1-4_D12 is shown in SEQ ID NO: 48, andits light-chain variable region sequence is shown in SEQ ID NO: 49.

(1-4) Assessment of Adenosine- and ATP-Binding Activity by CompetitivePhage ELISA

Based on the structures of 5′-Adenosine-PEG-biotin and ATP-PEG-biotin,there remained the possibility that clone ATNLSA1-4_D12 (heavy chainvariable region, SEQ ID NO: 48; light chain, SEQ ID NO: 49), which wasdemonstrated by the result of phage ELISA to have the ability to bind toboth 5′-Adenosine-PEG-biotin and ATP-biotin, recognizes the biotin tagor PEG moiety. Thus, to demonstrate that ATNLSA1-4_D12 is not anantibody that recognizes the biotin tag or PEG, whether the antigenbinding is inhibited by adenosine or ATP was tested by phage ELISA usingATNLSA1-4_D12, and hIL-6R-binding clone PF1 (heavy chain, SEQ ID NO: 50;light chain, SEQ ID NO: 51) prepared as a negative control.ATNLSA1-4_D12 and PF1 were each diluted with TBS and subjected to ELISAby the following procedure.

A StreptaWell 96 microtiter plate (Roche) was coated at room temperaturefor one hour with 100 μl of TBS containing biotin-labeled antigens (amixture of 5′-adenosine-PEG-biotin and ATP-PEG-biotin). After theantigens were removed by washing each well of the plate with TBST, thewells were blocked with 250 μl of 2% skim milk/TBS for one hour or more.2% skim milk/TBS was removed, and then the prepared, purified phageswere added to each well. The plate was allowed to stand at roomtemperature for one hour to allow binding of the antibody-displayingphages to the antigens in each well. Then, TBS that does not containantigen or that contains serial dilutions of ATP from an equal amount upto 10000 times that of the antigen was added to the wells. For thecompetition of the immobilized antigen with ATP, the plate was allowedto stand at room temperature for one hour. Then, after TBST wash, anHRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) dilutedwith TBS was added to each well. The plate was incubated for one hour.Following TBST wash, the TMB single solution (ZYMED) was added to eachwell. The chromogenic reaction in the solution of each well wasterminated by adding sulfuric acid. Then, the developed color wasassessed by measuring absorbance at 450 nm.

The measurement result is shown in FIG. 42. It was demonstrated that thehigher the ATP concentration, the smaller the degree of colordevelopment for ATNLSA1-4_D12 in the presence of an excess amount ofATP. Thus, the binding between ATNLSA1-4_D12 and its antigen wasdemonstrated to be inhibited in an ATP concentration-dependent manner.Meanwhile, in a control experiment with PF1 as a negative control, itsantigen binding was not detected regardless of the ATP concentration.The above finding demonstrates that ATNLSA1-4_D12 is an antibody thathas the ability to bind to ATP but does not recognize the biotin tag orPEG.

(1-5) Expression and Purification of Antibodies that Bind to ATP andAdenosine

The gene encoding the variable region of ATNLSA1-4_D12 was inserted intoan animal expression plasmid for human IgG1/Lambda. The antibody wasexpressed using the method described below. Cells of human fetal kidneycell-derived FreeStyle 293-F (Invitrogen) were suspended at a celldensity of 1.33×10⁶ cells/ml in FreeStyle 293 Expression Medium(Invitrogen) and aliquoted at 3 ml into each well of a 6-well plate. Theconstructed plasmid was introduced into the cells by lipofection. Afterfour days of culture in a CO₂ incubator (37° C., 8% CO₂, 90 rpm), theantibody was purified from the culture supernatants by a method known tothose skilled in the art using rProtein A Sepharose™ Fast Flow (AmershamBiosciences). Absorbance of the purified antibody solutions was measuredat 280 nm using a spectrophotometer. From the values obtained by themeasurement, the concentration of the purified antibody was calculatedusing an extinction coefficient determined by the PACE method (ProteinScience (1995) 4, 2411-2423).

(1-6) Assessment of the ATP- and Adenosine-Binding Antibody for its ATPand Adenosine Binding by Surface Plasmon Resonance

Biacore T200 (GE Healthcare) was used to analyze the interaction of D12,in which the constant region of IgG is linked to the variable region ofclone ATNLSA1-4_D12 with ATP- and adenosine-binding activity, inantigen-antibody reaction. Sensor chip CM5 or CM4 (GE Healthcare) wasimmobilized with an appropriate amount of protein A (Life technologies)by amine coupling. The antibody of interest was captured by the chip toallow interaction with ATP (Wako), adenosine (Wako), or ADP (adenosinediphosphate) (Wako) as an antigen. The running buffer used was 50 mMTris-HCl (Takara, T903), 500 mM NaCl, 0.01% (w/v) Tween20. The antigenwas allowed to interact for 30 seconds at a flow rate of 30 μl/min, andwas dissociated for 30 seconds. The interaction with the antigen wasassessed at 15° C. The antigen was diluted using the same runningbuffer.

The dissociation constant K_(D) (M) was calculated based on theassociation rate constant ka (l/Ms) and dissociation rate constant kd(l/s), both of which are kinetic parameters calculated from thesensorgram obtained by the measurement. Alternatively, the dissociationconstant KD (M) was calculated using steady state analysis. Eachparameter was calculated using the Biacore T200 Evaluation Software (GEHealthcare).

To calculate the K_(D) for adenosine, the binding response was assessedat various concentrations of adenosine in the presence or absence of 20μmol/l ADP. In addition, the binding response was separately assessed inthe presence of 20 μmol/l ADP. The response (R) for specific adenosinebinding was obtained by subtracting the value of binding response in thepresence of ADP alone from the binding response to variousconcentrations of adenosine in the presence of ADP, and then subtractingthe resultant value, which is assumed to correspond to the non-specificbinding components, from the value of binding response to adenosine inthe absence of ADP. From a curve in which adenosine concentration isplotted on the X axis and R calculated according to Formula 2 is plottedon the Y axis, the value of KD for adenosine was determined by the leastsquares method using the Solver function of Office Excel 2007(Microsoft).

R=Rmax×conc/(K _(D)+conc)

In Formula 2, conc represents adenosine concentration (mol/1) while Rmaxrepresents the value of response expected for the maximal binding ofadenosine to antibody. Measured response values were extracted by usingScrubber2 (BioLogics. Inc).

The KD of D12 determined by the measurement described above was 8.5μmol/l for ATP, 0.25 μmol/l for ADP, or 1100 μmol/l for adenosine. Thisresult demonstrates that D12 has binding activity to ATP, ADP, andadenosine; and it also suggests that D12 has binding activity to AMP(adenosine monophosphate) and cAMP (cyclic adenosine monophosphate).

Reference Example 2 Design of Library Using Anti-ATP/AdenosineAntibodies to Prepare ATP/Adenosine Switch Antibodies

In cancer tissues and inflamed tissues, not only the adenosine but alsothe ATP concentration is known to be high. Thus, it is beneficial to useantibodies for which both adenosine and ATP (referred to asATP/adenosine in this Example) can serve as a switch (specifically,antibodies that can bind to antigens when adenosine or ATP is present ata high concentration) as well as antibodies for which either adenosineor ATP alone serves as a switch. ATNLSA1-4_D12 described in ReferenceExample 1-4 is an antibody that binds to ATP/adenosine. As shown in FIG.43, ATP/adenosine is thought to be fit between the antibody and itstarget antigen, and thus the antibody comprises an antibody variableregion that comes in contact with the target antigen. Thus, the presentinventors conceived that synthetic antibody libraries that can isolateATP/adenosine switch antibodies whose binding activity to arbitraryantigens is altered depending on the presence of ATP/adenosine could beconstructed by collecting, as a library, antibody variable regionsegments that are capable of establishing contact with a target antigenand maintaining ATP/adenosine binding.

The crystal structure of the complex of ATP and ATP/adenosine antibodyATNLSA1-4_D12 obtained from a human antibody library as described inReference Example 1-4 was analyzed. The result of crystal structureanalysis revealed the mode of adenosine (or ATP) recognition by theantibody as well as identification of amino acid residues that areconsidered not to be substantially involved in adenosine (or ATP)binding in the antibody variable region. Amino acid residues that havebeen identified to be closely involved in the adenosine (ATP) bindingare Ser52, Ser52a, Arg53, Gly96, Leu100a, and Trp100c (Kabat numbering)in the heavy chain.

In designing such a library, sites that meet at least one of theconditions described below were selected as suitable for the libraryconstruction.

Condition 1: sites that are not greatly involved in ATP binding, or ifinvolved in the binding, a position having an amino acid other than thewild-type sequence that does not inhibit the ATP binding;

Condition 2: sites having a certain level of diversity of amino acidoccurrence frequency as repertoire of human antibodies; and

Condition 3: sites that are not essential for the formation of canonicalstructure.

In regions contained in both heavy chain and light chain of theATNLSA1-4_D12 sequence and that meet the conditions described above,amino acids in the CDR1 and CDR2 regions that have an occurrencefrequency of 2% or more in the germ line, as well as amino acids in theCDR3 region that have an occurrence frequency of 1% or more in the germline were comprehensively substituted. These substitutions were combinedto construct multiple variants of ATNLSA1-4_D12.

Alteration sites in the heavy chain (in the Table, positions indicatedby “Kabat” according to Kabat numbering), as well as amino acids beforealteration (in the table, amino acids referred to as “natural sequence”)at the sites and amino acids after alteration (in the table, amino acidsreferred to as “altered amino acids”) are shown in Table 46.

TABLE 46 HCDR1 HCDR2 HCDR3 Kabat 31 32 35 55 57 58 96 97 99 100 100aNative sequence T Y N N I N G R G D L Altered A A A A A A A A A amino Cacid E E D D D D D D D G G G G F F F F I I H H H H K K K K K M M L L N NN N N N N Q P S S S S S S S S S S R R R R R T T T T T W W V V V V V Y YY Y Y Y Y

Alteration sites in the light chain (in the Table, positions indicatedby “Kabat” according to Kabat numbering), as well as amino acids beforealteration (in the table, amino acids referred to as “natural sequence”)at the sites and amino acids after alteration (in the table, amino acidsreferred to as “altered amino acids”) are shown in Table 47.

TABLE 47 LCDR1 LCDR2 LCDR3 Kabat 26 27 27a 27b 27c 28 29 31 32 50 51 5253 54 55 89 90 91 92 93 94 95a 96 97 Native sequence T S S D V G G N Y EV S K R P S S Y A G S N V V Altered A A A A A A A A A A A A A amino Cacid E E E E E D D D D D D D D D D D D D D D G G G G G G G G G F F F F FI I I I I I H H H H H K K K K K K K K M M L L L L L L L N N N N N N N NN N N N Q Q Q Q Q Q P P P S S S S S S S S S S S S R R R R R R R R T T TT T T T T T T T T T T T W W W V V V V V V Y Y Y Y Y Y Y Y Y

Each variant expressed and purified by the method described in ReferenceExample 1-1 was assayed for its ATP and adenosine binding by the samemethod as described in Reference Example 1-6 using Biacore. Based on theassay result, the affinity of each variant for ATP was calculated as aKD value. Sites in the heavy chain, where alteration does not reduce theATP-binding ability to less than ⅕ of the binding ability ofATNLSA1-4_D12 (specifically, where the KD value is lower than 42.5μmol/l), and sites in the light chain where the ATP-binding ability islarger than that of ATNLSA1-4_D12 (specifically, where the KD value issmaller than 8.5 μmol/l), were assessed to be plausible for alteration.Amino acids substituted at those sites were judged to be appropriate forinclusion in the library (flexible residues to be introduced intolibrary).

Based on the assessment result on the ATP-binding ability of eachvariant, the ATP-binding ability was predicted to be reduced bycollecting each site to construct a library. Thus, substitutions wereintroduced at sites close to positions that are expected to be involvedin ATP binding, and various variants resulting from combination of thesesubstitutions were comprehensively assessed to test whether it ispossible to identify alterations which are expected to have effect ofaugmenting the ATP-binding ability. Such alteration sites (positionsindicated by “Kabat” according to Kabat numbering in the Table), andamino acids before alteration (amino acids referred to as “wild typesequence” in the table) and amino acids after alteration (amino acidsreferred to as “altered amino acids” in the table) at the sites areshown in Table 48.

TABLE 48 HCDR1 HCDR2 HCDR3 LCDR3 Kabat 33 50 56 95 98 100b 95 Nativesequence T S Y F K N N Altered A A A A A A A A amino C acid E E E E E EE D D D D D D D G G G G G G G F F F F F F F I I I I I I I I H H H H H HH K K K K K K K M M M M M M M M L L L L L L L L N N N N Q Q Q Q Q Q Q PP P P P P P S S S S S S R R R R R R R T T T T T T W W W W W W W W V V VV V V V V Y Y Y Y Y Y Y

Each variant expressed and purified by the method described in ReferenceExample 1-1 was assayed for its ATP and adenosine binding by the sameassay method using Biacore as described in Reference Example 1-6. Theassay result showed that the ATP and adenosine binding was expected tobe augmented by alterations at positions 56 and 100 and such accordingto Kabat numbering (for example, amino acid alteration such as Tyr56Hisand Asn100bLeu). It was determined that amino acids substituted at thesites could be included in a library (flexible residues to be introducedinto a library).

In the CDR regions of ATNLSA1-4_D12, amino acid repertoires containingamino acids selected by the above-described variant analysis as suitableto be included in a library (flexible amino acid residues to beintroduced into a library) and amino acids before alteration of theamino acids (specifically, amino acids included in the natural sequenceof ATNLSA1-4_D12), and sites comprising such repertoires were designedto construct a library for preparation of ATP/adenosine switchantibodies. The library was constructed in such a manner that in anamino acid repertoire the amino acid occurrence frequency is the samefor every amino acid (for example, when there are ten types of aminoacids in an amino acid repertoire, each amino acid occurs at 10%).

Sites comprising amino acid repertoires in the heavy chain (positionsindicated by “Kabat” according to Kabat numbering in the Table) andamino acid repertoires at the sites are shown in Table 49. Sitescomprising amino acid repertoires in the light chain (positionsindicated by “Kabat” according to Kabat numbering in the Table) andamino acid repertoires at the sites are shown in Table 50.

TABLE 49 HCDR1 HCDR2 HCDR3 Kabat 31 32 35 55 56 57 58 59 95 97 98 99 100100a 100b Native sequence T Y N N Y I N Y F R K G D L N Altered A 17%25% 11% 5% amino C acid E 9% 5% D 13% 9% 5% G 33% 17% 13% 11% 9% 5% F33% 13% 50% 9% 5% I 25% 11% 5% H 33% 50% 50% 13% 11% 9% 5% 17% K 25% 11%33% 9% 5% M 11% 5% 17% L 50% 11% 33% 5% 17% 50% N 50% 17% 13% 9% 5% 50%Q 9% 5% P 5% S 33% 17% 13% 5% R 17% 25% 11% 33% 9% 5% 17% T 33% 17% 13%5% W 9% 5% 17% V 11% 5% Y 33% 50% 13% 50% 50% 9% 5% 17%

TABLE 50 LCDR1 LCDR2 LCDR3 Kabat 27a 29 50 51 54 90 91 92 93 94 95 95a96 97 Native sequence S S E V R S Y A G S N N V V Altered A 17% 17% 14%13% 6% 17% amino C acid E 14% 17% 6% D 17% 14% 14% 13% 6% 11% G 17% 14%25% 14% 13% 6% 11% 17% F 17% 6% I 14% 13% 6% 11% H 6% 11% K 14% 50% 14%6% M 6% 17% L 25% 6% 11% 33% 17% N 25% 14% 13% 6% 11% O 14% 6% 11% P 6%33% S 50% 17% 14% 25% 17% 14% 13% 6% 11% 17% R 50% 14% 17% 13% 6% T 50%17% 25% 25% 14% 17% 14% 13% 6% W 14% 6% V 25% 25% 14% 33% 17% Y 14% 14%17% 14% 6% 11%

The result of sequence analysis suggests that the framework ofATNLSA1-4_D12 was derived from germ line VH3-21. Then, for the purposeof improving antibody stability, the framework sequence of ATNLSA1-4_D12was restored to the germ line sequence VH3-21 by introducing into theframework sequence of ATNLSA1-4_D12, alterations Gln01Glu, Gln05Val,Asp10Gly, Asn30Ser, Leu48Val, and Asn58Tyr (numerals represent Kabatnumbers). ATNLSA1-4_D12 variants expressed and purified by the methoddescribed in Reference Example 1-1 were measured for their Tm by DSC.DSC measurement was carried out by a method known to those skilled inthe art. Tm of the variant which results from adding these alterationsto ATNLSA1-4_D12 was markedly improved from 74.37° C. to 81.44° C., andstabilization of the structure was observed. It is sometimes preferableto use highly stable frameworks for antibody libraries, and thus aframework sequence to which alterations described above had been addedwas used as the framework sequence of a library. The framework used forthe library is shown in Table 51.

TABLE 51 SEQ ID Framework NO: Sequence Heavy chain 52 EVQLVESGGDLVKPGframework 1 GGLRLSCAASGFTFS Heavy chain 53 WVRQAPGKGLEWVS framework 2Heavy chain 54 RFTISRDNAKNSLYLQ framework 3 MNSLRAEDTAVYYCAR Heavy chain55 WGQGTLVTVSS framework 4 Light chain 56 QSALTQPPSAS framework 1GSPGQTVTISC Light chain 57 WYQQHPGKAPKLMIY framework 2 Light chain 58GVPDRFSGSKSGNTAS framework 3 LTVSGLQAEDEADYFC Light chain 59 GFFFTKLTVLframework 4

Genes were synthesized to comprise respective sequences in a librarydesigned as described above (DNA2.0), and the gene library was amplifiedwith primers that are capable of amplifying VH and VL respectively, byusing a collection (library) of the respective genes as a template. Thesequences of primers used for VL amplification are shown in SEQ ID NOs:81 and 82, while the sequences of primers used for VH amplification areshown in SEQ ID NOs: 83 and 84. The amplified rationally designed genelibrary of the heavy-chain and light-chain variable regions of humanantibody was inserted into an appropriate phagemid vector carrying botha human IgG-derived CH1 sequence and a human IgG-derived light chainconstant region sequence. The phagemid vector was introduced into E.coli by electroporation to construct a rationally designed library whichpresents Fab domains containing a human antibody variableregion-constant region, and from which one can isolate antibodies thatare capable of binding to antigens via adenosine or ATP as a switch.Such a rationally designed library which is constituted with various Hchains and L chains that have adenosine- or ATP-binding activity isexpected to be useful as a library containing human antibodies that,with the adenosine (or ATP) is fit in between antibody and antigen asshown in FIG. 43, can efficiently obtain adenosine/ATP switch antibodiesagainst any arbitrary antigen. Furthermore, as described above, sinceATNLSA1-4_D12 binds not only to adenosine and ATP but also to ADP, itwas predicted to have binding activity to AMP and cAMP which arestructurally similar to ATP, ADP, and adenosine. This suggests that suchlibraries are useful for isolating switch antibodies whose bindingactivity to arbitrary target antigens is altered depending on thepresence of any one or more small molecules of ATP, ADP, AMP, cAMP, andadenosine.

Reference Example 3 Acquisition of Antibodies that Bind to Antigens inthe Presence of Adenosine and ATP from Antibody Library Using PhageDisplay Techniques (3-1) Acquisition of Antibodies that Bind to Antigensin the Presence of Small Molecules Using a Mixture of Adenosine and ATPfrom Library

Antibodies that exhibit antigen-binding activity in the presence ofadenosine and/or ATP were obtained from the constructed phage-displaylibrary of rationally designed antibodies. To obtain antibodies, phagesdisplaying antibodies that exhibit the ability to bind to antigenscaptured by beads in the presence of adenosine and ATP were collected,and then the phages were collected in eluate from the beads in theabsence of adenosine and ATP.

Phages were produced in E. coli containing the phagemid vectorconstructed for phage display. To the culture medium of E. coli in whichphage production was carried out, 2.5 M NaCl/10% PEG was added toprecipitate phages. The precipitated phage fraction was diluted with TBSto prepare a library suspension. Then, BSA was added at a finalconcentration of 4% to the phage library suspension. Panning wasperformed using antigen-immobilized magnetic beads. The magnetic beadsused were NeutrAvidin coated beads (Sera-Mag SpeedBeadsNeutrAvidin-coated) and Streptavidin coated beads (Dynabeads M-280Streptavidin). Biotinylated human IL-6 receptor was used as an antigen.

500 pmol of biotin-labeled antigen and a final concentration of 1 mMATP-Na and adenosine were each added to the prepared phage librarysuspension. The phage library suspension was contacted with the antigen,adenosine, and ATP at room temperature for 60 minutes. The BSA-blockedmagnetic beads were added to the phage library suspension, and theantigen-phage complex was allowed to bind to the magnetic beads at roomtemperature for 15 minutes. The beads were washed once with ATP- andadenosine-dissolved TBS. Then, the beads were combined with 0.5 ml of 1mg/ml trypsin. Immediately after the beads were suspended at roomtemperature for 15 minutes, a phage suspension was collected from thebeads that were separated using a magnetic stand. The collected phagesuspension was added to 10 ml of E. coli cells of strain ER2738 at thelogarithmic growth phase (OD600=0.4 to 0.7). The E. coli was incubatedat 37° C. with gentle stirring for one hour to be infected by phage. Theinfected E. coli was seeded in a 225 mm×225 mm plate. Then, phages werecollected from the culture medium of the seeded E. coli to prepare aliquid stock of phage library.

The first round of panning was carried out to collect phages that arecapable of antigen binding in the presence of adenosine and ATP, whilethe second and subsequent rounds of panning were performed to enrichphages that are capable of antigen binding only in the presence ofadenosine and ATP. Specifically, 40 pmol of biotin-labeled antigen and afinal concentration of 1 mM adenosine and ATP were each added to theprepared phage library suspension. Thus, the phage library was contactedwith antigen, adenosine, and ATP for 60 minutes at room temperature.BSA-blocked magnetic beads were added, and the antigen-phage complex wasallowed to bind to the magnetic beads for 15 minutes at roomtemperature. The beads were washed with 1 ml of adenosine andATP-dissolved TBST (hereinafter referred to as (adenosine+ATP)/TBST),adenosine, and adenosine and ATP-dissolved TBS (hereinafter referred toas (adenosine+ATP)/TBS). Then, the beads were combined with 0.5 ml ofTBS. Immediately after the beads were suspended at room temperature, aphage suspension was collected from the beads separated using a magneticstand. After this treatment was repeated, the two separately elutedphage suspensions were combined together. The pIII protein (helperphage-derived protein pIII) that does not display Fab was cleaved offfrom phages by adding 5 μl of 100 mg/ml trypsin to the collected phagesuspension to eliminate the ability of phages that do not display Fab toinfect E. coli. The phages collected from the trypsinized phagesuspension were added to 10 ml of E. coli cells of strain ER2738 at thelogarithmic growth phase (OD600=0.4 to 0.7). The E. coli was incubatedat 37° C. with gentle stirring for one hour to be infected by phage. Theinfected E. coli was seeded in a 225 mm×225 mm plate. Then, phages werecollected from the culture medium of the seeded E. coli to prepare aphage library suspension. Panning was performed three times to isolateantibodies that have antigen-binding activity in the presence ofadenosine and ATP.

(3-2) Acquisition of Antibodies that Bind to Antigens in the Presence ofAdenosine and ATP Using a Negative Selection Method from AntibodyLibrary

A phage-display library of rationally designed antibodies was screenedfor antibodies that exhibit antigen-binding activity in the presence ofadenosine and/or ATP. As a first step of screening, the phage-displayantibody library was contacted with biotin-labeled antigen-streptavidinin the absence of adenosine and ATP to eliminate phages displayingantibodies that have antigen-binding activity even in the absence ofadenosine and ATP. Then, panning was performed in the same manner in thepresence of adenosine and ATP to screen for antibodies that exhibitantigen-binding activity in the presence of adenosine and ATP.

Phages were produced in E. coli containing the constructed phage-displayphagemid. To the culture medium of E. coli in which phage productiontook place, 2.5 M NaCl/10% PEG was added to precipitate phages. Theprecipitated phage fraction was diluted with TBS to prepare a librarysuspension. Then, BSA was added at a final concentration of 4% to thephage library suspension. Panning was performed usingantigen-immobilized magnetic beads. The magnetic beads used wereNeutrAvidin coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) andStreptavidin coated beads (Dynabeads M-280 Streptavidin).

Together with 250 pmol of biotin-labeled antigen, a mixture of adenosineand ATP was added at a final concentration of 1 mM to the prepared phagelibrary suspension. Thus, the phage library suspension was contactedwith the antigen, adenosine, and ATP for 60 minutes at room temperature.Then, BSA-blocked magnetic beads were added to the phage librarysuspension, and allowed to bind to the antigen-phage complex at roomtemperature for 15 minutes. The beads were washed once with(adenosine+ATP)/TBS. Then, the beads were combined with 0.5 ml of 1mg/ml trypsin solution. Immediately after the beads were suspended atroom temperature for 15 minutes, a phage suspension was collected fromthe beads that were separated using a magnetic stand. The collectedphage suspension was added to 10 ml of E. coli cells of strain ER2738 atthe logarithmic growth phase (OD600=0.4 to 0.7). The E. coli wasincubated at 37° C. with gentle stirring for one hour to be infected byphage. The infected E. coli was seeded in a 225 mm×225 mm plate. Next,M13KO7 Helper Phage (Takara Bio Inc.) or M13 KO7ΔpIII (calledHyperphage; PROGEN Biotechnik GmbH) was added to the culture solution ofthe seeded E. coli for infection, and after an overnight culture at 30°C., phages were collected from the supernatant to prepare a phagelibrary suspension displaying monovalent antibodies and a phage librarysuspension displaying multivalent antibodies.

The first round of panning was carried out to collect phages that arecapable of binding in the presence of adenosine and ATP, while thesecond and subsequent rounds of panning were performed to enrich phagesthat are capable of antigen binding only in the presence of adenosineand ATP. Specifically, 250 pmol of biotinylated antigen was added toBSA-blocked Sera-Mag NeutrAvidin beads, and allowed to bind at roomtemperature for 15 minutes. The beads were washed three times with TBS.The phage library suspension subjected to BSA blocking was added to thewashed beads, and allowed to bind at room temperature for one hour.Phages that did not bind to the antigens or beads were collected byisolating the beads using a magnetic stand. Forty pmol of biotin-labeledantigen, and a final concentration of 1 mM adenosine and ATP were eachadded to the collected phages. Thus, the phage library was contactedwith the antigen, adenosine, and ATP for 60 minutes at room temperature.Then, BSA-blocked magnetic beads were added to the mixture of thelabeled antigen, adenosine, ATP, and phage library, and allowed to bindto the antigen-phage complex for 15 minutes at room temperature. Thebeads were washed with 1 ml of (adenosine+ATP)/TBST and(adenosine+ATP)/TBS. Then, 0.5 ml of 1 mg/ml trypsin solution was addedto the mixture. After the mixed suspension was stirred at roomtemperature for 20 minutes, phages were collected from the beads thathad been separated using a magnetic stand. The collected phages wereadded to 10 ml of E. coli cells of strain ER2738 at the logarithmicgrowth phase (OD600=0.4 to 0.7). The E. coli was incubated at 37° C.with gentle stirring for one hour to be infected by phage. The infectedE. coli was seeded in a 225 mm×225 mm plate. Panning was performed threetimes to isolate antibodies that have antigen-binding activity in thepresence of adenosine and ATP.

(3-3) Assessment of Binding Activity in the Presence or Absence ofAdenosine and/or ATP by Phage ELISA

Phage-containing culture supernatants were collected according to aconventional method (Methods Mol. Biol. (2002) 178, 133-145) from singlecolonies of E. coli obtained by the method described above. Thecollected culture supernatants were treated by ultrafiltration usingNucleoFast 96 (MACHERY-NAGEL). 100 μl of the culture supernatants wereadded to each well of NucleoFast 96, and it was centrifuged (4500 g for45 minutes) to remove flow through. After addition of 100 μl of H₂O, theNucleoFast 96 was washed by centrifugation (4500 g for 30 minutes).Finally, 100 μl of TBS was added, and the NucleoFast 96 was allowed tostand for five minutes at room temperature. A phage suspension wascollected from the supernatant in each well of the NucleoFast 96.

The purified phages, to which TBS or (adenosine+ATP)/TBS was added, weresubjected to ELISA by the following procedure. A StreptaWell 96microtiter plate (Roche) was coated overnight with 100 μl of TBScontaining biotin-labeled antigen. After the antigen was removed bywashing each well of the plate with TBST, the wells were blocked with250 μl of 2% skim milk/TBS for one hour or more. 2% skim milk/TBS wasremoved, and then the prepared, purified phages were added to each well.The plate was allowed to stand at 37° C. for one hour to allow bindingof antibody-displaying phages to the antigen in each well in thepresence or absence of adenosine and/or ATP. After washing with TBST or(adenosine+ATP)/TBST, HRP-conjugated anti-M13 antibody (AmershamPharmacia Biotech) diluted with TBS or (adenosine+ATP)/TBS was added toeach well. The plate was incubated for one hour. Following washes withTBST or (adenosine+ATP)/TBST, the TMB single solution (ZYMED) was addedto each well. The chromogenic reaction in the solution of each well wasterminated by adding sulfuric acid. Then, the developed color wasassessed by measuring absorbance at 450 nm. The result revealed multipletypes of antibodies that bind human IL-6 receptors in the presence ofATP or adenosine. The result of phage ELISA is shown in Table 52.

TABLE 52 Number of panning rounds 3 4 Number of clones subjected toELISA 96 96 Number of positive clones (S/N ratio >10) 23 64 Number ofswitch clones (SM +/− ratio >2) 22 64 Number of switch clone sequences17 35

(3-4) Sequence Analysis of Switch Antibodies Whose Antigen-BindingActivity Varies Depending on the Presence or Absence of Adenosine andATP

Using specific primers (SEQ ID NOs: 79 and 80), genes were amplifiedfrom clones judged to have antigen-binding activity under the conditionwhere adenosine or ATP is present based on the phage ELISA resultsdescribed in Reference Example (3-3). The nucleotide sequences wereanalyzed, and based on the result, following clones were obtained andjudged to have binding activity to biotin-labeled hIL-6R in the presenceof adenosine or ATP: 6RAD2C1-4_001, 6RAD2C1-4_005, 6RAD2C1-4_011,6RAD2C1-4_026, 6RAD2C1-4_030, 6RAD2C1-4_042, 6RAD2C1-4_076,6RDL3C1-4_085, and 6RDL3C5-4_011 (Table 53).

TABLE 53 Clone name Heavy chain SEQ ID NO Light chain SEQ ID NO6RAD2C1-4_001 SEQ ID NO: 60 SEQ ID NO: 61 6RAD2C1-4_005 SEQ ID NO: 62SEQ ID NO: 63 6RAD2C1-4_011 SEQ ID NO: 64 SEQ ID NO: 65 6RAD2C1-4_026SEQ ID NO: 66 SEQ ID NO: 67 6RAD2C1-4_030 SEQ ID NO: 68 SEQ ID NO: 696RAD2C1-4_042 SEQ ID NO: 70 SEQ ID NO: 71 6RAD2C1-4_076 SEQ ID NO: 72SEQ ID NO: 73 6RDL3C1-4_085 SEQ ID NO: 74 SEQ ID NO: 75 6RDL3C5-4_011SEQ ID NO: 76 SEQ ID NO: 77

Reference Example 4 Acquisition of Antibodies that Bind to Antigens inthe Absence of Adenosine or ATP from Antibody Library UsingPhage-Display Techniques (4-1) Acquisition of Antibodies Whose AntigenBinding is Inhibited in the Presence of Small Molecules Using a Mixtureof Adenosine and ATP from a Library

Antibodies that bind to target antigens in the presence of smallmolecules serving as a switch were obtained as described in ReferenceExample 3 above. In this Reference example, the present inventorsattempted to obtain antibodies that bind to target antigens in theabsence of small molecules.

Antibodies that exhibit antigen-binding activity in the absence ofadenosine and/or ATP but whose binding ability is impaired in thepresence of adenosine and/or ATP were obtained from a phage-displaylibrary of rationally designed antibodies constructed in ReferenceExample 2. As a first step to isolate antibodies, a phage-displaylibrary of antibodies was contacted with biotinylated adenosine andbiotinylated ATP to collect a phage-display library of antibodies thatbind to adenosine and/or ATP. Then, the phage-display antibody librarywas contacted with biotinylated antigen-streptavidin in the absence ofadenosine and ATP to collect antibodies that bind to antigens in theabsence of adenosine and ATP. Panning was performed in the alternatingmanner described above to screen for antibodies that have bindingactivity to both antigen and adenosine and/or ATP. In the presence ofadenosine and ATP, the antigen binding of antibodies with suchproperties was expected to be inhibited by binding of adenosine and/orATP to the antibodies.

Phages were produced in E. coli containing the phagemid vectorconstructed in Reference Example 2 for phage display. To the culturemedium of E. coli in which phage production was carried out, 2.5 MNaCl/10% PEG was added to precipitate phages. The precipitated phagefraction was diluted with TBS to prepare a phage library suspension.Then, BSA was added at a final concentration of 4% to the phage librarysuspension. Panning was performed using antigen-immobilized magneticbeads. The magnetic beads used were NeutrAvidin-coated beads (Sera-MagSpeedBeads NeutrAvidin-coated) and Streptavidin-coated beads (DynabeadsM-280 Streptavidin).

500 pmol of biotinylated ATP, 2′-adenosine-PEG-Biotin, and5′-adenosine-PEG-Biotin were added to the prepared phage librarysuspension. Thus, the phage library suspension was contacted withadenosine and ATP for 60 minutes at room temperature. Then, BSA-blockedmagnetic beads were added to the phage library suspension and thecomplex of phage with adenosine and/or ATP was allowed to bind to themagnetic beads at room temperature for 15 minutes. The beads were washedonce with TBS, and then 0.5 ml of a 1 mg/ml trypsin solution was addedto the beads. Immediately after the beads were suspended at roomtemperature for 15 minutes, a phage suspension was collected from thebeads that were separated using a magnetic stand. The collected phagesuspension was added to 10 ml of E. coli cells of strain ER2738 at thelogarithmic growth phase (OD600=0.4 to 0.7). The E. coli was incubatedwith gentle stirring at 37° C. for one hour to be infected by phage. Theinfected E. coli was seeded in a 225 mm×225 mm plate. Then, phages werecollected from the culture medium of the seeded E. coli to prepare aphage library suspension.

The second round of panning was performed to enrich phages capable ofbinding to the biotinylated antigen in the absence of adenosine and ATP.Specifically, 250 pmol of biotinylated antigen was added to the preparedphage library suspension. Thus, the phage library suspension wascontacted with the antigen for 60 minutes at room temperature. Then,BSA-blocked magnetic beads were added to the phage library suspension,and the antigen-phage complex was allowed to bind to the magnetic beadsat room temperature for 15 minutes. The beads were washed twice withTBST and once with TBS. Then, 0.5 ml of a 1 mg/ml trypsin solution wasadded to the beads. Immediately after the beads were suspended at roomtemperature for 15 minutes, a phage suspension was collected from thebeads that were separated using a magnetic stand. The collected phagesuspension was added to 10 ml of E. coli cells of strain ER2738 at thelogarithmic growth phase (OD600=0.4 to 0.7). The E. coli was incubatedwith gentle stirring at 37° C. for one hour to be infected by phage. Theinfected E. coli was seeded in a 225 mm×225 mm plate. Then, phages werecollected from the culture medium of the seeded E. coli to prepare aphage library suspension.

At subsequent odd-numbered rounds, panning was performed in the samemanner as the first-round panning. However, the number of bead washeswith TBST and TBS was increased to three times and twice, respectively.

At subsequent even-numbered rounds, panning was performed in the samemanner as the second-round panning. However, in the fourth andsubsequent rounds of panning, the biotinylated antigen was reduced to 40pmol, and the number of bead washes with TBST and TBS was increased tothree times and twice, respectively.

(4-2) Assessment of Binding Activity in the Presence of Small Moleculesby Phage ELISA

Culture supernatants containing phages were collected according to aconventional method (Methods Mol. Biol. (2002) 178, 133-145) from singlecolonies of E. coli obtained by the method described above. Thecollected culture supernatants were ultrafiltrated using NucleoFast 96(MACHEREY-NAGEL). 100 μl of the culture supernatants were added to eachwell, and the NucleoFast 96 was centrifuged (4500 g for 45 minutes) toremove flow-through. 100 μl of H₂O was added to each well, and again theNucleoFast 96 was washed by centrifugation (4500 g for 30 minutes).After 100 μl of TBS was added, the NucleoFast 96 was allowed to stand atroom temperature for five minutes. Finally, a phage suspension wascollected from the supernatant in each well.

Purified phages, to which TBS, or ATP and adenosine/TBS had been added,were subjected to ELISA by the following procedure. A StreptaWell 96microtiter plate (Roche) was coated overnight with 100 μl of TBScontaining a biotin-labeled antigen. After the antigen not bound tostreptavidin was removed from each well of the plate by washing withTBST, the wells were blocked with 250 μl of 2% skim milk/TBS for onehour or more. 2% skim milk/TBS was removed, and then the prepared,purified phages were added to each well. The plate was allowed to standat 37° C. for one hour to allow binding of antibody-displaying phages toantigens in each well in the presence or absence of 10 mM adenosine andATP. After washing with TBST or 10 mM (ATP and adenosine)/TBST, anHRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) dilutedwith TBS or 10 mM (ATP and adenosine)/TBS was added to each well. Theplate was incubated for one hour. Following wash with TBST or 10 mM (ATPand adenosine)/TBST, the TMB single solution (ZYMED) was added to eachwell. The chromogenic reaction in the solution of each well wasterminated by adding sulfuric acid. Then, the developed color wasassessed by measuring absorbance at 450 nm.

Phage ELISA was carried out using 96 isolated clones to obtain from thelibrary of rationally designed antibodies, clone “I6RLSA1-6_011”, whichhad an antigen-binding activity to human IL-6 in the absence of ATP andadenosine, clone “HSADSA1-6_020”, which had antigen-binding activity tohuman serum albumin (HSA) in the absence of ATP and adenosine, as wellas clones “6RRLSA1-6_037” and “6RRLSA1-6_045”, which had anantigen-binding activity to human IL-6 receptor in the absence of ATPand adenosine (FIGS. 44, 45, and 46).

(4-3) Sequence Analysis of Antibodies for which Adenosine and ATP Serveas a Switch

Genes were amplified using specific primers (SEQ ID NOs: 79 and 80) fromclones that had been assessed to have antigen-binding activity in theabsence of adenosine and ATP based on the result of phage ELISAdescribed in (4-2). The nucleotide sequences of the genes were analyzed.Based on the analysis result, the amino acid sequences are shown inTable 54 below.

TABLE 54 Clone name Heavy chain SEQ ID NO Light chain SEQ ID NOI6RLSA1-6_011 138 139 HSADSA1-6_020 140 141 6RRLSA1-6_037 142 1436RRLSA1-6_045 144 145

Reference Example 5 Preparation of Biotinylated Human IgA-Fc

Among the naturally-occurring human IgA sequence, the Fc portion wasused as a human IgA (human IgA-Fc). To attach biotin to the C terminusof human IgA-Fc, a gene fragment encoding a specific sequence (AviTagsequence, SEQ ID NO: 147) for attaching biotin by biotin ligase waslinked via a linker. A gene fragment encoding a protein (SEQ ID NO: 146)in which human IgA-Fc is linked to AviTag sequence was inserted into avector expression in animal cells. The constructed plasmid vector wasintroduced into FreeStyle293 cells (Invitrogen) using 293Fectin(Invitrogen). In this experiment, genes for expressing EBNA1 (SEQ ID NO:148) and biotin ligase (BirA, SEQ ID NO: 149) were co-introduced, andbiotin was added for biotin-labeling the human IgA-Fc. Cells introducedwith the genes by the procedure described above were cultured at 37° C.under 8% CO₂ for six days to secrete the protein of interest in theculture supernatant.

The cell culture medium containing the biotinylated human IgA-Fc ofinterest was filtered through a 0.22-μm bottle-top filter to obtain theculture supernatant. The culture supernatant was diluted with 20 mMTris-HCl, pH 7.4 and applied to HiTrap Q HP (GE Healthcare) equilibratedwith 20 mM Tris-HCl, pH 7.4. The biotinylated human IgA-Fc of interestwas eluted with a concentration gradient of NaCl. Then, the HiTrap Q HPeluate was diluted with 50 mM Tris-HCl, pH 8.0, and applied to aSoftLink Avidin column (Promega) equilibrated with 50 mM Tris-HCl, pH8.0. The biotinylated human IgA-Fc of interest was eluted with 5 mMbiotin, 150 mM NaCl, 50 mM Tris-HCl, pH 8.0. Then, aggregates asunintended impurities were removed by gel filtration chromatographyusing Superdex200 (GE Healthcare), to obtain purified biotinylated humanIgA-Fc in which the buffer is replaced with 20 mM Histidine-HCl, 150 mMNaCl, pH 6.0.

INDUSTRIAL APPLICABILITY

Antigen-binding domains or antigen binding molecules comprising antigenbinding domains of the present invention whose antigen-binding activityvaries depending on the concentration of a small molecule compound, andpharmaceutical compositions containing them, have no systemic effect innormal tissues and blood but act in a reversible manner in cancer or atinflamed sites, which are the sites of pathology in target tissues,exert their medicinal effect while avoiding adverse effects, and enabletherapeutic treatment of diseases caused by target tissues. In case anantibody whose antigen binding is regulated depending on an unnaturalcompound is obtained, such antibody is highly useful because it can becontrolled by administration of exogenous compounds that activate theactivity or pharmaceutical effect of the antibody or exogenous compoundsthat can be administered in a non-invasive manner.

Moreover, various antigen-binding molecules useful for treatingtissue-specific diseases can be efficiently and rapidly obtained byusing a library that contains multiple antigen-binding domains orantigen-binding molecules comprising antigen-binding domains of thepresent invention whose sequences are different from one another andwhose antigen-binding activity varies depending on the concentration ofsmall molecule compounds.

1. A library that comprises mainly: (i) a plurality of antigen-bindingdomains or antigen-binding molecules comprising an antigen-bindingdomain, which have different sequences from one another; or (ii) nucleicacids that encode the plurality of antigen-binding domains orantigen-binding molecules comprising an antigen-binding domain, whichhave different sequences from one another; wherein the aforementionedantigen-binding molecule domains or antigen-binding molecules areantigen-binding domains or antigen-binding molecules comprising anantigen-binding domain whose antigen-binding activity varies dependingon the concentration of a small molecule compound.
 2. The library ofclaim 1, which is produced by a method comprising the steps of: (a)identifying amino acid sites that fulfill any one or more of (i) to(iii) below in antigen-binding domains whose antigen-binding activityvaries depending on the concentration of a small molecule compound or inantigen-binding domains that have binding activity to a small moleculecompound: (i) one or more amino acid sites that are not involved in thebinding to the small molecule compound; (ii) one or more amino acidsites that show diversity of amino acid occurrence frequency in theantibody repertoire of the animal species to which the parentantigen-binding domain belongs; and (iii) one or more amino acid sitesthat are not important for canonical structure formation; and (b)designing a library that comprises nucleic acids encoding unmodifiedantigen-binding domains/molecules, and nucleic acids that encodeindividually a plurality of variants of the aforementionedantigen-binding domains or antigen-binding molecules comprising anantigen-binding domain which have different sequences from one anotherand have modifications at one or more of the amino acid sites identifiedin step (a).
 3. The library of claim 2 which is produced by a methodcomprising the steps of: (a) identifying amino acid sites that fulfillany one or more of (i) to (iii) below in antigen-binding domains whoseantigen-binding activity varies depending on the concentration of asmall molecule compound or in antigen-binding domains that have bindingactivity to a small molecule compound: (i) one or more amino acid sitesthat are not involved in the binding to the small molecule compound;(ii) one or more amino acid sites that show diversity of amino acidoccurrence frequency in the antibody repertoire of the animal species towhich the parent antigen-binding domain belongs; and (iii) one or moreamino acid sites that are not important for canonical structureformation; (b) producing a plurality of variants of the aforementionedantigen-binding domains or antigen-binding molecules comprising anantigen-binding domain, which have different sequences from one anotherand have modifications at one or more of the amino acid sites identifiedin step (a); (c) identifying one or more amino acid modifications thatdo not substantially change the binding activity of each of theaforementioned variants to the small molecule compound; and (d)producing a library comprising nucleic acids that encode unmodifiedantigen-binding domains/molecules, and nucleic acids that encode aplurality of variants of the aforementioned antigen-binding domains orantigen-binding molecules comprising an antigen-binding domain, whichhave different sequences from one another and have one or more of theamino acid modifications identified in step (c).
 4. The library of claim1 produced by a method comprising the steps of: 1) contacting a librarycomprising a plurality of antigen-binding molecules having bindingactivity to a small molecule compound with the small molecule compound;and 2) concentrating from the library, nucleic acids that encode aplurality of variants of antigen-binding molecules having bindingactivity to the small molecule compound.
 5. The library of claim 4,wherein the aforementioned antigen-binding molecules are antigen-bindingmolecules that comprise heavy-chain variable regions and light-chainvariable regions of an antibody, and wherein the library is produced bya method comprising any one of the steps of: 1) designing a library byconcentrating nucleic acids that encode a plurality of variants ofantigen-binding molecules having binding activity to a small moleculecompound from the library of claim 4 which comprises nucleic acidsencoding one or more variants produced by modifying amino acidspositioned in the heavy chain variable regions; 2) designing a libraryby concentrating nucleic acids that encode a plurality of variants ofantigen-binding molecules having binding activity to a small moleculecompound from the library of claim 4 which comprises nucleic acidsencoding one or more variants produced by modifying amino acidspositioned in the light chain variable regions; and 3) designing alibrary by combining the antigen-binding molecule-encoding nucleic acidsconcentrated from each of the variable region libraries of steps 1) and2).
 6. The library of any one of claims 1 to 5, wherein theaforementioned antigen-binding molecules are fusion polypeptides formedby fusing an antigen-binding domain with at least a portion of a viruscoat protein.
 7. The library of any one of claims 1 to 5, wherein theaforementioned antigen-binding molecules are antigen-binding moleculescomprising antibody heavy chains and light chains, and the libraryfurther comprises a step of designing a synthetic library of the heavychains and/or light chains.
 8. The library of claim 7, wherein theantibody heavy chains and/or light chains comprise a germline-derivedframework sequence.
 9. The library of any one of claims 1 to 8, whereinthe aforementioned small molecule compound is a target tissue-specificcompound or an unnatural compound.
 10. The library of any one of claims1 to 9, wherein the aforementioned target tissue is a cancer tissue oran inflammatory tissue.
 11. The library of claim 10, wherein the cancertissue-specific compound is at least one compound selected from thegroup consisting of nucleosides that have a purine ring structure, aminoacids and their metabolites, lipids and their metabolites, primarymetabolites from sugar metabolism, and nicotinamide and its metabolites.12. The library of any one of claims 1 to 11, wherein the small moleculecompound is kynurenine, adenosine, adenosine monophosphate, adenosinediphosphate, or adenosine triphosphate.
 13. The library of any one ofclaims 1 to 12, wherein the amino acid sites not involved in bindingwith the small molecule compound are sites other than any one or more ofthe amino acids selected from below: H chain: 97, 100c, 101, 94, 95,100d, 100e, 33, 50, 52, 56, 57, 58, 99, 100, 100a, 54, 55 (KabatNumbering); and L chain: 49, 55, 95c, 96, 95a, 95b (Kabat Numbering).14. A method for producing an antigen-binding molecule comprising anantigen-binding domain whose antigen-binding activity varies dependingon the concentration of a small molecule compound, which comprises thesteps of: (a) contacting the library of any one of claims 1 to 13 withan antigen in the absence of a small molecule compound; (b) selecting anantigen-binding domain that does not bind to the antigen in step (a)above; (c) contacting the antigen-binding domain selected in step (b)above with the antigen in the presence of the small molecule compound;(d) selecting the antigen-binding domain that binds to the antigen instep (c) above; (e) linking the polynucleotide that encodes theantigen-binding domain selected in step (d) above with a polynucleotidethat encodes a polypeptide comprising an Fc region; (f) culturing a cellintroduced with a vector in which the polynucleotide obtained in step(e) above is operably linked; and (g) collecting the antigen-bindingmolecule from the culture solution of the cell cultured in step (f)above.
 15. A method for producing an antigen-binding molecule comprisingan antigen-binding domain whose antigen-binding activity variesdepending on the concentration of a small molecule compound, whichcomprises the steps of: (a) contacting the library of any one of claims1 to 13 with an antigen in the presence of a small molecule compound;(b) collecting an antigen-binding domain by dissociating it using thesmall molecule compound at a lower concentration than in step (a) above;(c) linking the polynucleotide that encodes the antigen-binding domaincollected in step (b) above with a polynucleotide that encodes apolypeptide comprising an Fc region; (d) culturing a cell introducedwith a vector in which the polynucleotide obtained in step (c) above isoperably linked; and (e) collecting the antigen-binding molecule fromthe culture solution of the cell cultured in step (d) above.
 16. Themethod of claim 14 or 15 for producing an antigen-binding molecule thatcomprises an antigen-binding domain whose antigen-binding activityvaries depending on the concentration of a small molecule compound,which further comprises the steps of: (a) contacting the library of anyone of claims 1 to 13 with a small molecule compound; and (b) selectingantigen-binding domains collected in step (a) above.
 17. The method ofany one of claims 14 to 16 for producing an antigen-binding molecule,wherein the small molecule compound is kynurenine, adenosine, adenosinemonophosphate, adenosine diphosphate, or adenosine triphosphate.
 18. Anantigen-binding molecule comprising an antigen-binding domain whoseantigen-binding activity varies depending on the concentration of anunnatural compound.
 19. A pharmaceutical composition that comprises theantigen-binding molecule of claim 18.